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. 2013 Oct 18;154(12):4873–4884. doi: 10.1210/en.2013-1325

Cross-Species Withdrawal of MCL1 Facilitates Postpartum Uterine Involution in Both the Mouse and Baboon

Chandrashekara Kyathanahalli 1, Jason Marks 1, Kennedy Nye 1, Belinda Lao 1, Eugene D Albrecht 1, Graham W Aberdeen 1, Peter W Nathanielsz 1, Pancharatnam Jeyasuria 1, Jennifer C Condon 1,✉
PMCID: PMC3836074  PMID: 24140717

Abstract

A successful postpartum involution permits the postnatal uterus to rapidly regain its prepregnancy function and size to ultimately facilitate an ensuing blastocyst implantation. This study investigates the molecular mechanisms that govern the initiation of the involution process by examining the signaling events that occur as the uterus transitions from the pregnant to postnatal state. Using mouse and baboon uteri, we found a remarkable cross-species conservation at the signal transduction level as the pregnant uterus initiates and progresses through the involution process. This study originated with the observation of elevated levels of caspase-3 activation in both the laboring mouse and baboon uterus, which we found to be apoptotic in nature as evidenced by the concurrent appearance of cleaved poly(ADP-ribose) polymerase. We previously defined a nonapoptotic and potential tocolytic role for uterine caspase-3 during pregnancy regulated by increased antiapoptotic signaling mediated by myeloid cell leukemia sequence 1 and X-linked inhibitor of apoptosis. In contrast, this study determined that diminished antiapoptotic signaling in the postpartum uterus allowed for both endometrial apoptotic and myometrial autophagic episodes, which we speculate are responsible for the rapid reduction in size of the postpartum uterus. Using our human telomerase immortalized myometrial cell line and the Simian virus-40 immortalized endometrial cell line (12Z), we demonstrated that the withdrawal of antiapoptotic signaling was also an upstream event for both the autophagic and apoptotic processes in the human uterine myocyte and endometrial epithelial cell.

This study examines the molecular mechanisms that contribute to the postpartum uterine involution process in both the pregnant rodent and nonhuman primate uterus. During pregnancy the human uterus increases in size and weight, from 50 g at implantation to more than 1000 g at term (1), and this study examined the triggers that permit the postpartum uterus to regain its prepregnancy weight and function. In general, an increase in the mass of a tissue or organ is regulated positively through hyperplastic and hypertrophic events and negatively by atrophy, apoptosis, and autophagic episodes. The increase in uterine size during pregnancy has largely been explained by early hyperplastic events (2) accompanied by hypertrophy of the uterine myometrial compartment and an increase in collagen content across gestation (3, 4).

Rapid decreases in muscle size such as those seen in the involuting uterus have been demonstrated in other tissues to occur through the initiation of atrophy related events (5, 6). An increase in atrophy-related ubiquitin ligases (7, 8) have been associated with uterine smooth muscle involution in the postpartum period (9). However, other essential markers of the atrophy process such as cathepsin L, forkhead box protein 01, and metallothionein-1 remain unchanged. These observations suggest that the ubiquitin proteasomal degradation pathway is not regulating uterine atrophy but may be associated with the onset of uterine autophagic or apoptotic processes (10, 11) in the postpartum period. Uterine autophagic and apoptotic activation have previously been indicated by the observation of myometrial autophagic vacuoles (12) and elevated levels of caspase-3 (CASP3) activation in the postpartum uterus (13–15); however, the mechanisms that regulate these events are still unclear.

This current study was initiated with the observation of CASP3 activation in the involuting uterus, which never attained the highest levels found during pregnancy but was observed to be apoptotic in nature. This finding was in stark contrast to our previous observation of nonapoptotic CASP3 activation in the pregnant uterus across gestation (16). We speculated that apoptotic CASP3 activation in the postpartum uterus may play a role in facilitating the postpartum involution process. Our group has recently identified that increased antiapoptotic signaling by myeloid cell leukemia sequence 1 (MCL1) to be a critical factor in maintaining uterine CASP3 in a nonapoptotic state across gestation (16, 17). In this current study, MCL1 declined to barely detectable levels in the laboring and postpartum period in both the mouse and baboon uteri. Therefore, we speculated that this withdrawal of MCL1-mediated antiapoptotic signaling allowed for the increased CASP3-mediated apoptotic indices identified in the laboring and postpartum uteri.

Surprisingly, although MCL1 withdrawal was observed in both the myometrial and endometrial involuting postpartum compartments, immunohistochemical analysis revealed apoptotic action, as evidenced by increased levels of active CASP3 and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining, which was restricted primarily to the involuting endometrium. These observations suggested that myometrial involution is not mediated through an apoptotic process and prompted us to examine other mechanisms potentially used by the myometrium to achieve a successful postpartum involution. It has recently been demonstrated that the ablation of MCL1 is an early event, associated not only with the induction of apoptosis (18) but also with the activation of autophagic signaling (19, 20). Therefore, this study examined the hypothesis that the postpartum uterus uses MCL1 withdrawal to activate individual and compartmentalized autophagic and apoptotic events to respectively modulate the myometrial and endometrial postpartum involution processes.

Materials and Methods

Mouse studies

All animal studies were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. Timed-pregnant female CD-1 mice (6–8 wk old) were obtained from Charles River and housed according to Institutional Animal Care and Use Committee guidelines. Uterine tissues (n = 3 for each gestational time point) were harvested between 8:00 and 10:00 am on gestational day (E) 18, E19, and days 1 and 2 postpartum (pp). Laboring uterine samples were collected from pregnant mice at E19 upon delivery of the first pup (E19IL). At each gestational time point, the uterine horn was cleared of all embryonic material and/or implantation/placentation sites to ensure that the mouse uterine tissues collected were limited to tissues of maternal origin such as the intact endometrial and myometrial layers. For immunofluorescence analysis, the uteri were fixed in 4% paraformaldehyde overnight and subsequently embedded in paraffin blocks for sectioning. The remaining uterine tissue was washed in 1× PBS and flash frozen for subsequent protein analysis.

Baboon studies

E164 baboon myometrium isolated from the lower uterine segment was kindly provided by Professor Eugene Albrecht (University of Maryland, Baltimore, Maryland). Female baboons (Papio anubis) weighing 14–16 kg were housed individually in air-conditioned rooms under standardized conditions. The experimental protocol used in the present study was approved by the Institutional Animal Care and Use Committee of the University of Maryland School of Medicine. Baboons received high-protein monkey kibble and fresh fruit twice daily, vitamins daily, and water ad libitum. Three untreated control animals underwent cesarean delivery on day 164 of gestation. The cervix was unaffected and closed in all of these animals. Samples of lower uterine segment were dissected immediately and flash frozen for later protein extraction. All animals were cared for and used strictly in accordance with US Department of Agriculture regulations and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Term laboring and nonlaboring baboon myometrium isolated from the lower uterine segment was kindly provided by Professor Peter Nathanielsz (University of Texas Health Sciences Center at San Antonio). All procedures were approved by the University of Texas Health Sciences Center, San Antonio, Institutional Animal Care and Use Committee. Cesarean section hysterectomy under halothane general anesthesia was performed in four term baboons that were not in labor (term = 175–185 days gestational age [dga]). Uterine electromyographic leads had been previously sited in the baboons that underwent cesarean section hysterectomy to ascertain that these animals were not in labor. The cervix was uneffaced and closed in all of these animals. Electromyographic analysis for 48 hours preceding cesarean section hysterectomy revealed no contraction activity. Cesarean hysterectomy was also performed on four term animals in spontaneous labor when myometrial activity was in the form of high-amplitude short-lived contractions. Cervical dilation was occurring in all of these animals and the cervix was fully effaced. Samples of the lower uterine segment were dissected immediately and flash frozen for later protein extraction.

Human telomerase immortalized myometrial (hTERT) and 12Z cell cultures

The hTERT cell line was derived from premenopausal nonpregnant uterine tissue as previously described (21). The 12Z endometrial epithelial cell line was derived from primary cultures of endometriotic biopsies transfected with Simian virus-40 T antigen as previously described (22). Briefly, cells were cultured in DMEM/F12 (Gibco) with 10% fetal bovine serum (FBS; Gibco) and antibiotic/antimycotic (10 000 U/mL; Gibco) at 37°C in 95% air with 5% CO2. For each experiment, hTERT cells and 12Z cells were seeded at 1.5–2 × 106 cells per 10-cm2 tissue culture dish in phenol red-free DMEM/F12 with 10% charcoal-stripped FBS for 12 hours and subsequently serum starved for 12 hours prior to treatment with recombinant human Fas ligand (FasL) (catalog number 126-FL-010/CF) (R&D Systems) at 100 ng/mL in the presence of 10 μg/mL of a cross-linking antibody (MAB050; R&D Systems) for 16 hours in phenol red-free DMEM/F12 containing 0.5% charcoal-stripped FBS.

Extraction of cytoplasmic and nuclear proteins from frozen uterine tissues

Cytoplasmic and nuclear protein extracts were prepared from frozen uterine tissue as described previously (23). Protein disulfide isomerase (PDI) and nuclear receptor coactivator 3 (NCOA3) were used to ensure equal loading for immunoblotting and as markers to determine the purity of the isolated nuclear and cytoplasmic protein fractions, respectively. Subcellular protein concentrations were determined in triplicate by bicinchoninic acid protein assay using the Pierce BCA assay kit (ThermoScientific).

Extraction of cytoplasmic and nuclear protein fractions from the hTERT and 12Z cells

Isolation of cytoplasmic and nuclear extracts from cultured hTERT and 12Z cells were performed as described above and previously (23). Briefly, cells grown in monolayer were washed with PBS, scraped, and centrifuged at 3000 × g for 1 minute at 4°C. The pellet was homogenized in ice-cold nuclear protein extraction buffer 1 buffer containing 10 mM HEPES (pH 7.5), 10 mM MgCl2, 5 mM KCl, 0.1% Triton X-100, and 1× protease/phosphatase inhibitor cocktail (Roche) with a 23-guage needle. The remaining steps are as detailed previously (23).

Immunoblotting and densitometric analysis

Equal amounts of protein were separated on NuPAGE precast gradient gels (Life Technologies) and transferred to Hybond-P polyvinyl difluoride membranes (Millipore). The membranes were blocked in 5% nonfat milk prepared in 1× Tris-buffered saline with Tween 20 for 1 hour at room temperature and then incubated with the primary antibodies overnight. This was followed by incubation with horseradish peroxidase-conjugated secondary antibodies diluted in 5% milk-1× Tris-buffered saline with Tween 20 (TBS-T). Immunoreactive bands were visualized using an enhanced chemiluminescence detection system (Thermo Scientific). The sources of primary antibodies and their concentrations are as follows: PDI (#3501, 1:1000), cleaved CASP3 (#9664, 1:1000), cleaved poly(ADP-ribose) polymerase (PARP; # 9541, 1:750), X-linked inhibitor of apoptosis (XIAP; # 2042, 1:200), phosphorylated mammalian target of rapamycin (pMTOR; #2971, 1:500), phosphorylated total non-phosphorylated p53 (TP53) (#9284, 1:500) (all from Cell Signaling Technologies). Mouse uterine Light Chain (LC) I and II levels were detected using the cell signaling LC3A/B antibody (#12741, 1:500), which detects endogenous levels both the LC3 I and II. Unfortunately, this antibody was inefficient in the baboon tissues; therefore, a second LC3 antibody cell signaling antibody was used (#2775, 1:500), which detects endogenous levels of total LC3B protein and a stronger reactivity is observed with the type II form of LC3A/B (Santa Cruz Biotechnology). Antibodies for MCL1 (SC-819,1:200) and NCOA3 (PA1–845, 1:2000) procured from Santa Cruz Biotechnology and ThermoScientific respectively were used for the study. The immunoreactive cytoplasmic and nuclear bands obtained by immunoblotting were quantified using ImageJ (National Institutes of Health, Bethesda, Maryland) and normalized to PDI and NCOA3, respectively, because their protein concentrations were found to remain relatively unchanged in the pregnant mouse and baboon uterus across gestations and in the hTERT cell line across treatment conditions (17).

Immunofluorescence

Paraffin-embedded uterine tissues isolated from pregnant mice at E18, E19, day 1, and 2 pp were sectioned at 5-μm intervals and collected on Superfrost Plus slides (Fisher Scientific). Paraffin sections were deparaffinized through xylene and rehydrated through an alcohol series. Sections were blocked in 1× PBS containing 5% normal donkey serum and incubated in the primary antibody diluted in 1× PBS overnight at 4°C at the following concentrations: MCL1 (1:100), cleaved CASP3 (1:100; Cell Signaling); Bcl-2-associated X protein (BAX; number 2772, 1:250), LC3I/II (1:100), and MCL1 (1:100). The following day, sections were washed in 1× PBS and incubated in the fluorescent secondary antibody for 1 hour at 25°C. Donkey antirabbit secondary antibodies conjugated to either Alexa flour 488 or Cy3 (Jackson ImmunoResearch) were used at a dilution of 1:500 in PBS. The sections were washed and incubated with 4′, 6-diamidino-2-phenylindole (DAPI) to stain nuclei and mounted in gelvatol. Images were collected at a magnification of ×10 on a Leica DMRBE (Leica Microsystems) using a Q-Imaging Micro Publisher 5.0 RTV (QImaging). Nonspecific autofluorescence of abundant red blood cells in the E19IL tissue unfortunately made the immunohistochemical analysis for MCL1 and BAX uninterpretable. The antibody for XIAP was unsuitable for immunohistochemical analysis in our hands at each gestational time point.

Reverse transfection of hTERT cells with Mcl1 small interfering RNA (siRNA)

Silencer Select siRNA specific for MCL1 (siRNA identification s8583, control sense 5′-CCAGUAUACUUCUUAGAAATT-3′ and test antisense 5′-UUUCUAAGAAGUAUACUGGGA-3′ were obtained from Ambion) and were applied to hTERT and 12Z cells as previously described (17).

TUNEL staining

Paraffin-embedded uterine tissues were sectioned at 5-μm intervals and collected on Superfrost Plus slides (Fisher Scientific). Paraffin sections were deparaffinized through xylene and rehydrated through an alcohol series. Sections were permeabilized with 20 μg/mL Proteinase K for 8–10 minutes at room temperature and were stained with the DeadEnd Fluorometric TUNEL assay system as per the manufacturer's instructions (#G3250; Promega).

Statistical analysis

All data are representative of at least three individual experiments performed in triplicate. Immunoblots were analyzed by densitometry using ImageJ (National Institutes of Health). Statistical analysis of immunoblots was performed with StatPlus:mac software 2009 version (AnalystSoft Inc). Baboon and mouse gestational data points were subjected to a one-way ANOVA followed by pairwise comparison (Student-Neuman-Keuls method) to determine differences between groups. Values of P < .05 were considered significant.

Results

Increased apoptotic CASP3 activation in the laboring and postpartum mouse uterus

As can be seen in Figure 1A, cytoplasmic CASP3 activation as indicated by the appearance of a doublet at approximately 19 and 17 kDa was readily detectable at E18, absent at E19, but reappeared in the laboring and postpartum uterus (17 kDa fragment detectable of day 1 and 2 pp). To examine the apoptotic potential of the laboring and postpartum uterine CASP3, we investigated the incidence of cleaved PARP (24) in the nuclear proteins extracted from pregnant mice at E18, E19, 19IL, day 1, and 2 pp by Western blot analysis. As can be seen in Figure 1A, prior to term, the CASP3 activation was devoid of apoptotic potential as evidenced by the lack of cleaved PARP products. However, the increased PARP cleavage in the laboring and postpartum uteri indicates a successful initiation of the apoptotic signaling cascade.

Figure 1.

Figure 1.

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A, Increased activation of CASP3 and PARP cleavage in the laboring and postpartum mouse uterus. Cytoplasmic and nuclear uterine extracts were examined by Western blot analysis for the appearance of cleaved CASP3 and PARP, respectively, at E18, E19 (nonlaboring), 19IL (laboring), and 1 and 2 days pp (n = 3 for each gestational time point). B, Increased CASP3 activation associated with PARP cleavage in the laboring baboon uterus. Cytoplasmic and nuclear baboon uterine extracts were examined by Western blot analysis for the appearance of cleaved CASP3 and PARP, respectively, at E164 (n = 3), at term nonlaboring (n = 5), and term laboring (n = 4). PDI and NCOA3 both found at constant levels across each time point were used as loading controls for the cytoplasmic and nuclear fractions, respectively. These data are representative of at least three uterine samples per gestational time point and are represented as mean ± SEM. ROD, relative optical density. Data labeled with different letters are significantly different from each other (P < .05).

Increased apoptotic CASP3 activation in the term laboring baboon uterus

Cytoplasmic and nuclear extracts isolated from pregnant baboon uterine tissues at E164 and at term in both the laboring and nonlaboring state were examined for the presence of active CASP3 and PARP cleavage, respectively, by Western blot analysis. As observed in Figure 1B, significantly elevated levels of active CASP3 in the term laboring baboon uterus was associated with increased PARP cleavage. In contrast, active uterine CASP3 at E164 and in the term nonlaboring tissues demonstrate a nonapoptotic signature as indicated by diminished levels of PARP cleavage. These data indicate that analogous to the pregnant mouse (Figure 1A), baboon (Figure 1B) uterine CASP3 activation gains apoptotic potential with the onset of labor.

Decreased antiapoptotic signaling in the laboring and postpartum mouse uterus

Cytoplasmic extracts isolated from uterine tissues of pregnant mice at E18, 19, 19IL, day 1, and 2 pp examined by Western blot analysis confirmed an increase in the antiapoptotic molecules MCL1 and XIAP to term, which were significantly diminished in the laboring and postpartum uteri (Figure 2A).

Figure 2.

Figure 2.

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A, Elevated uterine levels of antiapoptotic signaling to term decrease in association with the onset of labor and the postpartum period. Cytoplasmic uterine extracts were examined for the antiapoptotic factors MCL1 and XIAP at E18, E19, 19IL, and days 1 and 2 pp. B, Elevated levels of the antiapoptotic factors MCL1 and XIAP in the term pregnant baboon uterus decline with the onset of labor. Cytoplasmic baboon uterine extracts were examined by Western blot analysis for the appearance of MCL1 and XIAP at E164 (n = 3), at term nonlaboring (n = 5), and term laboring (n = 4). PDI and NCOA3, both found at constant levels across each time point, were used as loading controls for the cytoplasmic and nuclear fractions, respectively. These data are representative of at least three uterine samples per gestational time point and are represented as mean ± SEM. ROD, relative optical density. Data labeled with different letters are significantly different from each other (P < .05).

Diminished MCL1 and XIAP in the laboring baboon uterus

Cytoplasmic uterine extracts isolated from the pregnant baboon at E164, term nonlaboring, and term laboring were examined for the presence of the antiapoptotic proteins MCL1 and XIAP by Western blot analysis. As can be seen in Figure 2B, both MCL1 and XIAP levels are undetectable at E164 but are highly elevated to term. However, a significant withdrawal of uterine XIAP and MCL1 occurs as the pregnant baboon uterus transitions to its laboring status, in an analogous manner to the diminished antiapoptotic signaling profile observed in the laboring and postpartum mouse uterus (Figure 2A). These data indicate that in both the pregnant mouse (Figure 2A) and baboon (Figure 2B), uterine antiapoptotic signaling peaks at term; however, upon transition to the laboring and postpartum state, antiapoptotic signaling is diminished, allowing for the onset of apoptotic CASP3 action as is indicated by increased PARP cleavage (Figure 1, A and B).

Elevated TUNEL staining in the laboring and postpartum mouse uterus

To examine the histological location of the increased apoptotic action of active uterine CASP3 in the laboring and postpartum mouse uteri, TUNEL staining was performed (25). As expected elevated levels of TUNEL staining were confined to the laboring and postpartum uteri, with very few positive cells found in the pregnant uterus at E18 and E19 (Figure 3). These data confirm that increased PARP cleavage products observed in Figure 1, A and B, in the laboring and postpartum uteri are likely as a result of increased apoptotic action of uterine CASP3. However, TUNEL staining was largely isolated to the laboring and postpartum endometrial compartment, with minor TUNEL staining observed in the pregnant uterus at E18 and E19 in either the myometrial and endometrial compartments. These data indicate that elevated levels of CASP3 and cleaved PARP (Figure 1, A and B) identified by Western blot in the laboring and postpartum uterus likely represent an endometrial apoptotic activation signature, which allows for the rapid remodeling of the endometrial layer of the laboring and postpartum uterus.

Figure 3.

Figure 3.

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Increased TUNEL staining was isolated to the endometrial compartment of the postpartum mouse uterus. Analysis of uterine paraffin sections isolated at E18, E19, and days 1 and 2 pp were examined for the appearance and the localization of TUNEL staining (green). E, endometrium; M, myometrium; DAPI, blue.

Elevated proapoptotic action in the mouse endometrium at term

Immunohistochemical analysis identified elevated MCL1 levels in both the myometrial and endometrial compartments of the pregnant mouse uterus at E18 and E19, which declined to barely detectable levels at days 1 and 2 pp. Proapoptotic BAX levels were undetectable at E18; however, elevated levels were isolated to epithelial cells of the endometrium and the glandular component of the endometrial layer of the E19 and day 1 pp mouse uterus (see Figure 4).

Figure 4.

Figure 4.

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Decreased MCL1 in the postpartum mouse uterus accompanied by increased BAX immunoreactivity. Immunofluorescence analysis of uterine paraffin sections isolated at E18, E19, and days 1 and 2 pp were examined for the appearance and the localization of BAX (red) and MCL1 staining (red). E, endometrium; M, myometrium; EG, endometrial glands; RBC, red blood cells; DAPI, blue.

Elevated endometrial TUNEL and CASP3 and myometrial LC3II in the postpartum mouse uterus

Uterine CASP3 is at its highest levels at E13 (26), and its isolation to the smooth muscle cell component of the myometrial compartment during pregnancy is confirmed (see Figure 5). We also verify that at E13 active CASP3 is nonapoptotic as is indicated by the lack of myometrial TUNEL staining. However, at day 1 pp, we observe a relocation of active CASP3 to the endometrial compartment, which is accompanied by elevated TUNEL staining, signifying the apoptotic nature of the postpartum endometrial CASP3. Moreover, we observed activation of the uterine autophagosome as defined by the appearance of LC3II on day 1 pp restricted to the myometrial compartment of the postpartum uterus. These data indicate that the endometrium uses apoptotic cell death, whereas the myometrium likely exploits autophagic signaling to successfully remodel the postpartum uterus to regain its prepregnancy size and function.

Figure 5.

Figure 5.

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An elevated myometrial autophagic response occurs in parallel with increased apoptotic CASP3 activation in the endometrial compartment in the postpartum mouse. Immunofluorescence analysis of uterine paraffin sections isolated at E13 and day 1 pp were examined and compared for the appearance and the localization of CASP3 (red), TUNEL staining (green), and LC3II (green). Hematoxylin and eosin (H&E) staining were used to identify gross uterine histological structure. E, endometrium; M, myometrium.

Up-regulation of autophagic signaling in the postpartum mouse uterus

To confirm the contribution of the autophagic process in postpartum uterine remodeling, uterine tissues of pregnant mice at E18, E19, 19IL, and days 1 and 2 pp were examined by Western blot analysis for the presence of the autophagic mediators, phospho-MTOR, phospho-TP53, and LC3I and II (see Figure 6A). Phospho-MTOR acts as an inhibitor of autophagy and is expressed at high levels at E18 and gradually declines to barely detectable levels in the postpartum uterus by day 2 pp (see Figure 6A). Phospho-TP53, the molecule that inhibits MTOR activation, also rises to maximal levels day 1 pp, indicating a further derepression of the autophagy process in the postpartum uterus. Additionally, a marked elevation in the conversion of LC3I to LC3II confirms an increase in the autophagic process on days 1 and 2 pp in the mouse uterus (see Figure 6A).

Figure 6.

Figure 6.

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A, Elevated p53 activates autophagy in the postpartum mouse myometrium through the down-regulation of MTOR signaling. Nuclear uterine extracts were examined by Western blot analysis for the appearance of phospho-TP53, and cytoplasmic uterine extracts were examined for the presence of phospho-MTOR and LC3I conversion to LC3II at E18, E19, 19IL, and days 1 and 2 pp (n = 3 for each gestational time point). B, Elevated p53 activates autophagy in term and laboring baboon myometrium through the down-regulation of MTOR signaling. Nuclear uterine extracts were examined by Western blot analysis for the appearance of phospho-TP53 and cytoplasmic uterine extracts were examined for the presence of phospho-MTOR and LC3I conversion to LC3II at E164 (n = 3), term nonlaboring (n = 5), and term laboring (n = 4). PDI and NCOA3 found at constant levels across each gestational time point were used as loading controls for the cytoplasmic and nuclear fractions, respectively. These data are representative of at least three uterine samples per gestational time point and are represented as mean ± SEM. ROD, relative optical density. Data labeled with different letters are significantly different from each other (P < .05).

Activation of the autophagic signaling response in the laboring baboon uterus

Analogous to the postpartum mouse uterus, we have identified a significant decline in phospho-MTOR levels associated with increased phospho-TP53 levels in the term and term laboring uterine tissues (see Figure 6B). Although a statistically significant increase in TP53 was observed (see Figure 6), variability was identified in phospho-TP53 levels at E164, which we speculate may be as a result of the variable length of gestation in the pregnant baboon, which may result in this sample being closer to term than the other E164 samples. Elevated levels of LC3II were also found in the term and term laboring tissues in comparison with the earlier gestational time point of E164. These data suggest both the pregnant baboon and mouse uteri initiate the uterine involution process during labor by up-regulating an autophagic response (see Figure 6, B and A). In an effort to normalize the laboring cohort, laboring uterine samples were collected when uteri exhibited high amplitude, short-lived contractions. We speculate that the variable time it took for the individual uteri to attain this phenotype may contribute to the variation between samples in the term laboring baboon myometrium. However, despite the observed variation, all analyses reached statistical significance (see Figures 1, 2, and 6).

MCL1 ablation in the hTERT cell and 12Z results in autophagic activation in the myometrial cell and apoptotic activation in the endometrial cell

Myometrial hTERT and endometrial 12Z cell depletion of MCL1 was mediated by siRNA transfection, and the cells were examined for markers of autophagic and apoptotic signaling (see Figure 7). Removal of MCL1 was found to initially result in the activation of autophagic pathway as indicated the appearance of increased LC3II levels in the myometrial hTERT cell line. However, the introduction of an apoptotic stimulus such as FasL resulted in the transformation of that autophagic signaling profile to an apoptotic phenotype as indicated by the appearance of cleaved CASP3 and PARP in the MCL1-depleted cells. In contrast, removal of MCL1 alone from the endometrial 12Z epithelial cell line was sufficient to initiate both an autophagic and a robust apoptotic response as indicated by increased CASP3 activation and PARP cleavage (see Figure 7B), which ultimately resulted in cell death in the presence or absence of FasL.

Figure 7.

Figure 7.

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Ablation of MCL1 induced an autophagic response in the human myometrial cell and an apoptotic response in the human endometrial cell. hTERT (A) and 12Z cells (B) were transfected with MCL1 siRNA for 24 hours and treated with or without FasL for 12 hours. Cytoplasmic extracts were analyzed form MCL1 protein levels by Western blot for the following experimental conditions; scrambled siRNA control, MCL1 siRNA, Fas plus scrambled siRNA, and FasL plus MCL1 siRNA. Cytoplasmic extracts from the same conditions were analyzed for phospho-T53, LC3I to LC3II conversion, CASP3, and PARP cleavage. PDI and NCOA3 were used as loading controls. These data are representative of three independent experiments (n = 3). FL, full length; CL, cleaved.

Discussion

In this current study, we have defined the role of MCL1 in regulating autophagic and apoptotic events that initiate postpartum remodeling in the myometrial and endometrial fractions of the laboring and postpartum uterus. We have observed a cross-species conservation of the signal transduction events between the mouse and the baboon uterus in vivo that allows for a rapid and efficient postpartum involution process. Using our hTERT cell line and the endometrial 12Z cell line, we identified that the apical role of MCL1 remained conserved in the human uterine myocyte and the human epithelial endometrial cell, in that it has the capacity to trigger both the autophagic and apoptotic cell fates in vitro. Our group has previously identified a role for antiapoptotic signaling mediated by MCL1 in the myometrial smooth muscle layers of the pregnant uterus (26). Gestationally regulated CASP3 activation in the pregnant mouse uterus was maintained in a nonapoptotic manner due to elevated levels of antiapoptotic signaling mediated by members of the B cell lymphoma 2 (BCL2) and the inhibitor of apoptosis families, MCL1, and XIAP (16, 17), allowing for the maintenance of uterine quiescence. The reappearance of CASP3 (Figure 1, A and B) in the laboring and postpartum uterus in both mouse and baboon initiated this current investigation into the role of apoptotic CASP3 and antiapoptotic MCL1 in the postpartum involution process.

CASP3 activity in the laboring and postpartum rodent and primate uterus was found to be associated with the onset of apoptosis as is indicated by the appearance of PARP cleavage products (Figure 1, A and B). We identified in both the mouse and baboon uterine models that the disappearance of the antiapoptotic signaling molecules MCL1 and XIAP from the laboring and postpartum uteri interrupted the protective antiapoptotic signaling network (Figure 2, A and B) and a surge in apoptotic BAX signaling at term (Figure 4) prompted an increase in uterine apoptotic CASP3 action (Figure 1, A and B). Immunohistochemical analysis revealed that active CASP3, once limited to the myometrial fraction of the pregnant mouse uterus, was now confined to the endometrial compartment of the postpartum mouse uterus (Figure 5). CASP3 was confirmed as the source of the uterine postpartum apoptotic signature by TUNEL analysis, which also colocalized to the postpartum endometrium (Figures 3 and 5). These data infer that postpartum remodeling of the endometrial compartment is achieved in an apoptotic fashion induced by increased apoptotic CASP3 action similar to that seen in the cycling endometrium during the late secretory and menstrual phase of the cycle (27–31).

Because CASP3 apoptotic action was limited to the endometrial fraction during the postpartum involution process, we investigated alternate mechanisms that may regulate postpartum myometrial involution. We examined the likelihood that postpartum myometrial involution was achieved through the autophagic process (32, 33). During autophagy LC3I is conjugated to phosphatidylethanolamine to form LC3II, which is recruited to autophagosomal membranes (34), and the appearance of LC3II is widely used for monitoring autophagy and autophagy-related processes (20).

We examined uteri of the mouse and baboon for evidence of a potential autophagic response during the laboring and postpartum phase. Immunohistochemical analysis initially identified elevated levels of LC3II isolated to the mouse myometrial compartment of the postpartum uterus (Figure 5), indicating that an autophagic response limited to the myometrial compartment may be responsible for the postpartum uterine myocyte involution process. MTOR signaling, which acts to inhibit the autophagic process (35), was found to decline towards term in both the pregnant mouse and baboon uteri (Figure 6, A and B). Moreover, phospho-TP53, which inhibits MTOR signaling, increased in the postpartum mouse uteri and the term baboon uteri (Figure 6, A and B). However, the unexpected withdrawal of phospho-TP53 observed on day 2 pp may be due to phospho-TP53 no longer being required for further MTOR suppression because MTOR is no longer present in the day 2 pp uterus. The opposing actions of TP53 and MTOR have previously been described, in which activation of TP53 inhibits MTOR activity and thus up-regulates its downstream targets, resulting in autophagy (36, 37). Therefore, we speculate that increased phospho-TP53 levels block uterine MTOR signaling, allowing for increased myometrial autophagic action, which was further confirmed by the up-regulation of LC3II levels in the postpartum mouse and laboring baboon uteri (Figure 6, A and B).

We next examined the mechanism regulating the parallel transformation of the nonapoptotic pregnant uterus into an actively apoptosing endometrium while also promoting an autophagic response in the myometrium during the postpartum period. It has previously been demonstrated in vitro and in vivo that inhibition of the antiapoptotic protein MCL1 is an early event in the induction of apoptosis but also regulates the activation of autophagy (19, 38). As we have demonstrated in Figure 2, A and B, MCL1 levels, as indicated by Western blot analysis, decline in the pregnant mouse and baboon uterus with the onset of labor. Immunohistochemical analysis of the mouse uterine tissues revealed that MCL1 was present in both the myometrial and endometrial layers of the pregnant uterus and confirmed its elimination from both compartments in the laboring and postpartum conditions (Figure 4). Others have recently demonstrated that after MCL1 withdrawal, autophagy is the major outcome unless proapoptotic signaling is concomitantly activated (19), allowing for an apoptotic rather than an autophagic response. Several investigators have identified that the apoptotic process is responsible for the morphological changes that occur to the endometrium during the secretory phase of the estrous cycle (39, 40). Specifically the proapoptotic molecule BAX has been shown to be expressed in human endometrial tissue (41) with elevated levels in the secretory apoptotic endometrium (42) and in the cytoplasm of the glandular epithelium of the endometrium in the human (30), monkey (43), and cat (44), resulting in an apoptotic endometrial profile (45).

Examination of BAX levels in the postpartum mouse uterus demonstrated elevated levels isolated to the endometrial component of the E19 mouse uterus and by day 1 pp was limited to the glandular epithelium of the endometrium, as shown in Figure 4. We suggest that the parallel activation of both autophagy and apoptosis in the involuting uterus is primarily dictated by the withdrawal of antiapoptotic signaling mediated by MCL1 in both the myometrium and the endometrium. MCL1 withdrawal from both compartments triggers a myometrial autophagic response; however, the concurrent increase in BAX signaling confined to the endometrium summons an apoptotic phenotype.

We speculate that a similar MCL1 withdrawal mediated mechanism may regulate the involution process in the human uterus as it transitions between the pregnant and postpartum state. Some women fail to undergo successful uterine involution in the postpartum period, and antiapoptotic signaling molecules and members of the BCL2 family (of which MCL1 is a member) have previously been implicated in problems at the uteroplacental junction during the human pregnancy (46, 47). With subinvolution (48, 49), women are at a high risk for excessive postpartum hemorrhage (50, 51). Persistence of antiapoptotic signaling has previously been associated with cases of subinvolution, and in the case of placental cretas, overexpression of antiapoptotic factors has been implicated (53, 53). In contrast, reduced levels of the antiapoptotic signaling have been associated with preeclampsia, inappropriate autophagy, and increased apoptosis (54).

To determine the role of antiapoptotic signaling in human uterine autophagic and apoptotic signaling, we inhibited MCL1 levels in the human myometrial hTERT cell line and the human endometrial 12Z cell line. Using MCL1 siRNA, we examined the potential of active MCL1 withdrawal to regulate both the autophagic and apoptotic processes in the absence and presence of an apoptotic stimuli. As can be seen in Figure 7A, inhibition of MCL1 alone did not cause an increase in apoptosis as measured by the lack of CASP3 activation or PARP cleavage in the hTERT cell. However, the onset of autophagy as indicated by increased LC3II levels was observed in the MCL1-depleted myometrial hTERT cell (Figure 7A). hTERT cells exposed to FasL alone resulted in increased CASP3 activation but remained protected against CASP3-mediated apoptotic cell death due to the antiapoptotic action of endogenous MCL1 levels (17). In contrast, cells devoid of MCL1 that were challenged with the proapoptotic stimuli, Fas ligand, failed to undergo autophagy as indicated by a lack of LC31 to LC3II conversion but underwent apoptotic cell death as can be seen by CASP3 activation, giving rise to PARP cleavage. These data confirm the capacity of upstream MCL1 depletion in the absence or presence of apoptotic stimuli to respectively activate the uterine autophagic and apoptotic responses in the human analogous to what was observed in the baboon and mouse myometrium. In contrast, the inhibition of MCL1 or the presence of FasL in the endometrial 12Z cell line was sufficient to initiate an autophagic and ultimately the apoptotic process as measured by increased CASP3 activation and PARP cleavage and cell death (Figure 7B). These data suggest that similar to our in vivo observations the myometrial and endometrial responses to MCL1 ablation differ in vitro. The uterine myocyte cell type undergoes a default autophagic response, whereas the endometrial epithelial cell type will ultimately resort to an apoptotic phenotype upon MCL1 removal.

This current study highlights how the withdrawal of uterine MCL1 is central to the molecular processes that trigger uterine involution as outlined in Figure 8, which we have found to be conserved at a cross-species level in the mouse and the baboon uterus. We argue that the gestationally regulated profile of MCL1 is likely critical in inhibiting the involution process and its withdrawal is a consequence of the onset of labor. Indeed, most women undergoing cesarean section are administered a uterotonic agent either oxytocin or misoprostol to promote the involution process and thereby limit the risk of postpartum hemorrhage (55). These data confirm that the signal, which we speculate to be MCL1, which inhibits the involution process from occurring during pregnancy, is not fetal but maternal in origin. Our previous analysis has defined a role for progesterone (P4) in the positive regulation of CASP3 activity in the pregnant mouse uterine myocyte across gestation (26), which prompts the question as to the role of P4 in regulating the increased CASP3 activity associated with endometrial remodeling postpartum. We speculate that declining progesterone receptor- and/or P4-mediated signaling at term may indirectly regulate local apoptotic and autophagic signaling through mediating a decline uterine MCL1 and/or XIAP levels. Understanding these molecular mechanisms that govern the postpartum involution process may afford us opportunities to develop therapeutic strategies to avoid uterine subinvolution in the postpartum process. Also, recognizing the gestationally regulated strategies that govern the timing of MCL1 withdrawal may provide answers to defining the factors that regulate the timing of labor.

Figure 8.

Figure 8.

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A model representing the central role of MCL1 in the regulation of endometrial apoptosis and myometrial autophagy in the postpartum uterine involution process.

Acknowledgments

We thank Caroline Rieser, BSc, and Mary Rebecca Moreci, BSc, for their critical reading of this manuscript.

This work was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development Grant 1R01HD065011 and the March of Dimes Grant 21FY12–152.

Disclosure Summary: The authors have nothing to disclose.

Footnotes

Abbreviations:
BAX
Bcl-2-associated X protein
BCL2
B cell lymphoma 2
CASP3
caspase-3
E
gestational day
FasL
Fas ligand
FBS
fetal bovine serum
hTERT
human telomerase immortalized myometrial
LC
Light Chain
MCL1
myeloid cell leukemia sequence 1
MTOR
mammalian target of rapamycin
NCOA3
nuclear receptor coactivator 3
P4
progesterone
PARP
poly(ADP-ribose) polymerase
PDI
protein disulfide isomerase
pMTOR
phosphorylated MTOR
pp
postpartum
siRNA
small interfering RNA
TP53
total non-phosphorylated p53
TUNEL
terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling
XIAP
X-linked inhibitor of apoptosis.

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