Uterine endoplasmic reticulum stress-unfolded proteinresponse regulation of gestational length is caspase-3and -7–dependentChandrashekara Kyathanahallia,b, Kenna Organc, Rebecca S. Morecic, Prashanth Anamthathmakulaa,b,Sonia S. Hassana,b,d, Steve N. Caritisc, Pancharatnam Jeyasuriaa,b,c,d, and Jennifer C. Condona,b,c,d,1

aDepartment of Obstetrics and Gynecology, School of Medicine, Wayne State University, Detroit, MI 48201; bDepartment of Physiology, School of Medicine,Wayne State University, Detroit, MI 48201; cDepartment of Obstetrics and Gynecology, Magee-Womens Research Institute, University of Pittsburgh,Pittsburgh, PA 15213; and dPerinatology Research Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda,MD 20892

Edited by John J. Eppig, The Jackson Laboratory, Bar Harbor, ME, and approved October 1, 2015 (received for review September 15, 2015)

We previously identified myometrial caspase-3 (CASP3) as apotential regulator of uterine quiescence. We also determinedthat during pregnancy, the functional activation of uterine CASP3is likely governed by an integrated endoplasmic reticulum stressresponse (ERSR) and is consequently limited by an increasedunfolded protein response (UPR). The present study examinedthe functional relevance of uterine UPR-ERSR in maintainingmyometrial quiescence and regulating the timing of parturition.In vitro analysis of the human uterine myocyte hTERT-HM cell linerevealed that tunicamycin (TM)-induced ERSR modified uterinemyocyte contractile responsiveness. Accordingly, alteration ofin vivo uterine UPR-ERSR using a pregnant mouse model signifi-cantly modified gestational length. We determined that “normal”gestational activation of the ERSR-induced CASP3 and caspase 7(CASP7) maintains uterine quiescence through previously uniden-tified proteolytic targeting of the gap junction protein, alpha 1(GJA1); however, surprisingly, TM-induced uterine ERSR triggeredan exaggerated UPR that eliminated uterine CASP3 and 7 tocolyticaction precociously. These events allowed for a premature in-crease in myometrial GJA1 levels, elevated contractile responsive-ness, and the onset of preterm labor. Importantly, a successfulreversal of the magnified ERSR-induced preterm birth phenotypecould be achieved by pretreatment with 4-phenylbutrate, a chaper-one protein mimic.

endoplasmic reticulum stress | caspase-3 and -7 | unfolded proteinresponse | preterm labor | uterus

Although the rates of preterm birth (PTB) continue to decreasein the United States, there has been a steady rise in prevalence

globally over the past decade (1). Multiple risk factors have beenassociated with preterm labor (2); however, the events that precedeand elicit the signals allowing for the onset of premature uterinecontractions and labor remain unclear. Thus, PTB continues topose an acute risk for neurodevelopmental and respiratory com-plications that adversely effect neonatal health (3, 4). In this study,we demonstrate that the pregnant rodent uterus uses an integratedunfolded protein response (UPR)-endoplasmic reticulum stressresponse (ERSR) pathway to maintain steady levels of activatedcaspase-3 (CASP3) and caspase-7 (CASP7), which preserve uterinequiescence across gestation. We also demonstrate that an increasein the adaptive UPR limits CASP3 and 7 activation to allow theinduction of both term and preterm labor mediated through in-creased levels of gap junction protein, alpha 1 (GJA1).We have identified that CASP3 and 7 play compensatory roles

in regulating uterine myocyte quiescence. Previous investigationsfrom our laboratory and others have identified a gestationallyregulated activation of nonapoptotic uterine CASP3 duringpregnancy (5–7). Furthermore, we have proposed that activationof CASP3 during pregnancy occurs as a result of gestationallyregulated increases in uterine ERSR (8).

The endoplasmic reticulum (ER) is the organelle that facilitatesprotein folding and transport (9), misfolded protein ubiquitination,and proteasomal degradation. Functional irregularities at the ERlevel cause the accumulation of misfolded proteins, leading to ini-tiation of an ERSR (10). A prolonged and/or excessive ERSR hasbeen implicated in potentiating increased CASP3 and 7 activation(11). In every pregnancy, the uterus experiences physiological andbiochemical stimuli that in other biological systems trigger anERSR, including stretch (12), inflammation (13), hormone fluctu-ations (14, 15), hypoxia (16), hyperplasia (17), hypertrophy (18),and demand for metabolic fuels (19).We propose that the pregnant uterus also may use the ERSR

to activate and harness the tocolytic potential of CASP3 and 7,allowing it to retain its quiescent phenotype during these periodsof adaptation. Furthermore, we propose that an inappropriateERSR or UPR mismanagement may modulate uterine CASP3and 7 activity, thereby influencing gestational length. We testedthis hypothesis by manipulating the uterine ERSR and UPR inthe pregnant mouse and monitoring the levels of active CASP3and 7 together with the timing of labor. We found that excessive/prolonged potentiation of uterine ERSR fails to maintain uterineCASP3 and 7 levels, owing to the unexpected triggering of a pre-cociously heightened adaptive UPR, which ultimately leads to theonset of PTB. Coadministration of 4-phenylbutrate (PB) andtunicamycin (TM) allows for the maintenance of uterine CASP3and 7 levels, which reverses and rescues the TM-induced PTBphenotype. We have identified GJA1, known to play an essential

Significance

Preterm birth is a leading cause of neonatal mortality, with apoorly understood etiology. The maintenance of uterine quies-cence across gestation is fundamental for term parturition. Atthe molecular level, an integrated uterine endoplasmic reticulumstress-unfolded protein response (UPR-ERSR) regulates bothcaspase-3 (CASP3) and caspase-7 (CASP7), preservingmyometrialquiescence throughout gestation. Here we show that prolongedERSR diminishes the tocolytic potential of uterine CASP3 and 7,causing an increase in myometrial contractile responsivenessand onset of preterm labor in pregnant mice. Prophylaxis with4-phenylbutrate, however, maintains active uterine CASP3 and 7and prolongs gestational length. This study establishes a criticalrole for UPR-ERSR in the regulation of pregnant uterine myocytequiescence.

Author contributions: P.J. and J.C.C. designed research; C.K., K.O., R.S.M., P.A., and J.C.C.performed research; S.N.C. and J.C.C. contributed new reagents/analytic tools; S.S.H., P.J.,and J.C.C. analyzed data; and J.C.C. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. Email: jcondon@med.wayne.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1518309112/-/DCSupplemental.

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role in myometrial gap junction intercellular communication (20–22), integrating the signals for active contraction during labor in thepregnant uterus, as a target of uterine CASP3 and 7 activity bothin vitro and in vivo.

ResultsTM Induces ER Stress and Activates Apoptotic CASP3 Action in HumanUterine Myocytes in Vitro. We examined the functional relevance ofUPR-ERSR modulation of uterine myocyte CASP3 and 7 activityby exposing the telomerase immortalized human uterine myocyte(hTERT-HM) cell line (23) to TM. The induction of UPR-ERSRwas measured by the abundance of DNA damage-inducible tran-script 3 (DDIT3), cleaved CASP3 and CASP7, poly-ADP ribosepolymerase (PARP) and the chaperone protein, glucose-regulatedprotein 78 kDa (GRP78). As seen in Fig. 1A, GRP78, which acts asa prosurvival adaptive component of the UPR, was up-regulated ateach TM exposure (0.5–5 μg/mL). Because the accumulation ofexcessive misfolded proteins demands additional GRP78, its limitedavailability can potentiate the onset of an ERSR-induced apoptoticsignaling cascade. Accordingly, prolonged or excessive exposure toTM (2.5 μg/mL for 48 h and 5 μg/mL for 6–48 h) resulted in robustinduction of DDIT3, cleaved CASP3 and CASP7 levels, andPARP inactivation.

GJA1 Is a Target Substrate for Uterine Myocyte CASP3 Activity inVitro. Using the hTERT-HM cell line, we also observed that ex-posure to TM (2.5 μg/mL for 48 h and 5 μg/mL for 6–48 h) resultedin diminished levels of GJA1 in the human uterine myocyte (Fig.1B). Using the web servers SitePrediciton and Cascleave, weidentified a CASP3 and 7 cleavage site at amino acid 339 with>99% specificity in the GJA1 protein sequence (Fig. S1) (24, 25).We used UV light exposure to activate CASP3 and 7 in an alter-nate non–ERSR-dependent manner in the hTERT-HM cells, andobserved increased levels of active CASP3 and 7, corresponding todecreased levels of GJA1. On CASP3 and 7 inhibition, GJA1 levelsremained elevated (Fig. 2).

Excessive ERS Negatively Regulates CASP3 Activation in PregnantMouse Uterus in Vivo. Using the pregnant mouse model, we injectedvehicle control or TM (0.04, 0.2, and 1mg/kg i.p.) at embryonic day (E)15. Uteri were isolated 24 h after TM administration and

monitored for activation of the UPR-ERSR. As shown in Fig.3A, a single i.p. injection of TM at 0.04 mg/kg did not elict auterine UPR-ERSR; however, TM administration at 0.2 mg/kgsucessfully potentiated the UPR-ERSR, as evidenced by increasedGRP78 and DDIT3 levels. Uterine myocyte CASP3 levelsremained unchanged, suggesting that augmented chaperone actionallowed resolution of the 0.2 mg/kg TM-induced ERSR. In con-trast, TM administered at 1 mg/kg demonstrated a divergent UPR-ERSR activation profile from our in vitro findings. An exaggeratedincrease in GRP78 levels, the consequent elimination of DDIT3,and diminished levels of active CASP3 resulted in a surge in GJA1levels (Fig. 3B). Other organs, such as the heart (Fig. S2A), did notexhibit modified UPR-ERSR/GJA1 levels in response to i.p.administered TM.

Excessive ERS Induces a PTB Phenotype in Pregnant Mice.We examinedthe timing of the onset of labor in pregnant mice administered asingle i.p injection of TM (0, 0.04, 0.08, 0.12, 0.2, and 1 mg/kg) atE15. Administration at <0.12 mg/kg did not alter gestationallength, and all mice delivered live births at term. Pregnant mice givenTM ≥0.12 mg/kg had a higher incidence of PTB with delivery ofnonviable neonates at E16 (TM 1.0 mg/kg) and E17 (TM 0.2 mg/kg)(Table 1). No maternal morbidity was observed in this study, andthe increased incidence of PTB occurred independently of a de-cline in circulating P4 levels (Fig. S3).

PB Attenuates TM-Induced ERSR in Human Uterine Myocytes in Vitro.We next investigated the ability of the chaperone mimic PB topromote an adaptive UPR response in the context of an excessiveTM-induced ERSR. For this, hTERT-HM cells were treated with5 μg/mL TM and 5 mM PB concurrently and/or individually. Asshown in Fig. 4, TM has the capacity to increase GRP78 andDDIT3 levels, increase CASP3 and 7 activation, and decreaseGJA1 levels. In contrast, pretreatment with PB resolved the TM-induced ERSR, thereby suppressing DDIT3 levels, resulting indiminished uterine myocte CASP3 and 7 activation and allowingGJA1 to remain at control levels. Quantitative RT-PCR analysisconfirmed the TM-induced changes in uterine myocyte GJA1levels at a posttranscriptional level (Fig. S4).

ERSR Activation of CASP3 Disrupts the Uterine Myocyte ContractileApparatus. Immunohistochemical analysis revealed that GJA1,normally located in the cell membrane, fails to traffic properly inthe presence of TM-induced ERSR. In contrast, cotreatment

Fig. 1. ERSR mediates CASP3 activation and consequent GJA1 decline in thehuman uterine myocyte in vitro. (A) hTERT-HM cells were treated with TM(0–5 μg/mL) for 0–48 h and examined for activation of the ERSR and UPR byimmunoblotting. GRP78 was up-regulated on TM exposure. Prolonged orexcessive exposure to TM (2.5 μg/mL for 48 h or 5 μg/mL 6–48 h) resulted inactivation of the ERSR indicated by an elevation in DDIT3, cleaved (CL)CASP3, CL CASP7, and CL PARP. (B) On CASP3 and 7 activation (2.5 μg/mL for48 h or 5 μg/mL for 6–48 h), diminished levels of GJA1 are observed. Arepresentative blot from three different experiments is shown.

Fig. 2. GJA1 is a substrate for active CASP3 in the human uterine myocytein vitro. Using UV light to activate apoptosis in the hTERT-HM cell line, weexamined GJA1 levels in the absence or presence of 100 μM QVD-OPH. In-hibition of CASP3 and CASP7 activation resulted in sustained GJA1 levels. Arepresentative blot from three individual experiments is shown.

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with PB and TM allows the resolution of the uterine myocteERSR and CASP3 and 7 activation (Fig. 4), permitting suc-cessful trafficking of GJA1 to the cell membrane similar tocontrol cells and cells treated with PB alone (Fig. 5A).

TM-Induced ERSR Modifies Uterine Myocyte Contractile Ability. Todetermine the basal contractile phenotype of the human uterinemyocyte in response to changing ERSR levels, we performed 3Dcollagen contraction assays with hTERT-HM cells treated withvehicle, 5 μg/mL TM, and 5 mM PB individually and/or con-currently. As shown in Fig. 5B, control and PB-treated cellscontracted at a similar rate; however, in the presence of TM,the uterine myocyte retained a noncontractile phenotype. Incontrast, hTERT-HM cells coexposed to PB and TM dem-onstrated increased contractile responsiveness similar to con-trol levels.

In Vivo Administration of PB Modulates Uterine CASP3 Activation andGJA1 Levels. To modulate the in vivo PTB consequences of theexaggerated ERSR induced by TM, we administered PB (50 mg/kgi.p.) to pregnant mice (E15) at 1 h before and 12 h after TM(1 mg/kg) exposure. PBS, PB (50 mg/kg), and TM (1 mg/kg) wereadministered i.p. individually and served as controls. As shown inFig. 6, TM administration in vivo induced an exaggerated UPR,resulting in increased GRP78 levels, causing a decline in CASP3and 7 activity and a dramatic increase in GJA1 levels. Co-administration of TM and PB promoted the maintenance ofCASP3, CASP7, and GJA1 at control levels. We previouslyidentified uterine alpha actin (ACTA2) and gamma actin(ACTG2) as targets of CASP3 (6), and in the present study, wehave confirmed their regulation in an ERSR- dependent mannerin vivo (Fig. S5).

Pretreatment of Pregnant Mice with PB Rescues the TM-Induced PTBPhenotype. Pregnant mice were pretreated with PB (50 mg/kg) at1 h before and 12 h after TM (0.2 and 1.0 mg/kg) exposure atE15 and then monitored for timing of labor. Table 2 shows thatpretreatment with PB rescued the TM (0.2 mg/kg)-ERSR–

induced PTB phenotype. In the animals exposed to higher doses ofTM (1 mg/kg), PB administration significantly delayed the onsetof preterm labor. Treatment with vehicle and PB alone deliveredlive pups at term. The PB-mediated rescue of the PTB pheno-type occurred independent of changes in uterine histone acety-lation (26), isoprenylation rate (27), and PR levels or ratios (28–30) (Fig. S6).

The Pregnant Uterine UPR-ERSR Is Regulated in a Progesterone- andProgesterone Receptor-Dependent Manner. Pregnant mice exposedto elevated levels of P4 (2 mg/d) from E13 to E19 demonstrated areduced UPR as term approached, resulting in an elevated and pro-longed uterine ERSR, increased CASP3 activation, and diminishedGJA1 levels (Fig. 7A). In contrast, treatment with the PR in-hibitor RU-486 resulted in a dramatic decline in DDIT3 levelsand a precocious surge in GRP78 levels, indicating resolution ofuterine ERSR, at 4 h after RU-486 administration. Rapidelimination of active CASP3 resulted in a premature surge inGJA1 levels (Fig. 7B). Because the hTERT-HM cell line used inthis study does not express detectable levels of PR, the action ofPR and P4 on regulation of the uterine myocyte ERSR in vitrowas undetectable (Fig. S7). In human pregnancy, similar to themouse model, term (39–42 wk gestation; n = 6) nonlaboringmyometrium demonstrated an elevation in the UPR, marked by

Fig. 3. Excessive ERS negatively regulates CASP3 activity in the pregnantmouse uterus in vivo. (A) ER homeostasis was maintained in the murineuterus after i.p. administration of TM 0.04 and 0.2 mg/kg. However, TM at1 mg/kg triggered increased GRP78 levels, decreased DDIT3 (Fig. S2B), anddiminished active CASP3 levels compared with controls (n = 6 per group).(B) Elevated GJA1 levels are observed in the pregnant mouse uterus at 24 hafter i.p. administration of TM (1 mg/kg). A representative blot from threedifferent experiments is shown. Statistical comparisons were done usingone-way ANOVA, followed by the Newman–Keuls multiple-comparison test.*P ≤ 0.05 compared with controls.

Table 1. Excessive uterine ERSR results in the onset of labor inTM-treated mice

TM, mg/kg Onset of delivery

0 E19 (3 of 3)0.04 E19 (3 of 3)0.08 E19 (3 of 3)0.12 E17 (1 of 3)0.20 E17 (3 of 6)1.0 E16 (6 of 6)

Mice at E15 exposed to TM <0.08 mg/kg delivered at term. TM exposure≥0.12 mg/kg resulted in the onset of PTB between 18 and 32 h. n = 6 pergroup.

Fig. 4. TM-induced ERSR can be modulated by increased chaperone actionin vitro. Administration of PB (5 mM) to the hTERT-HM prevents TM (5 μg/mL)-induced ERSR, as indicated by diminished DDIT3, CL CASP3 and 7, and CL PARPlevels, allowing for recovery of GJA1 to control levels. n = 3 per group.

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increased GRP78 levels compared with preterm nonlaboringpatients (32–34 wk gestation; n = 6) (Fig. S8).

Uterine CASP3 and 7 Have a Compensatory Role for Each Other WithRespect to Uterine Myocyte Contractility. hTERT-HM cells weretransfected either separately or together with validated siRNAsfor CASP3 and CASP7. Western blot analysis confirmed theknockdown of CASP3 and 7. Administration of TM (5 μg/mL)increased the activation of CASP7 in CASP3-deficient hTERT-HM cells, whereas active CASP3 levels were increased in cellstransfected with CASP7 siRNA. As expected, TM failed to ac-tivate either CASP3 or CASP7 in hTERT-HM cells transfectedwith both CASP3 and CASP7 siRNAs; however, GJA1 levelswere reduced in the presence of CASP3 and/or CASP7 (Fig. 8A).Using the aforementioned siRNA-treated hTERT-HM cells, weperformed collagen contraction assays in the absence or pres-ence of TM. As shown in Fig. 8B, the presence of either CASP3or CASP7 resulted in relaxation of the uterine myocyte collagenlattice. In contrast, ablation of both CASP3 and CASP7 allowedsuccessful contraction of collagen gel lattice similar to that seenin control cells.

DiscussionThe present study demonstrates that the pregnant uterine myocyteuses the UPR-ERSR to maintain uterine quiescence by hijackingthe tocolytic action of CASP3 and 7 generated in response to thecellular stresses experienced across gestation. We observed thatexaggerated, prolonged, or inappropriate uterine ER stress ulti-mately causes an imbalanced ERSR, precipitating CASP3 and 7modulation and resulting in the onset of PTB. Our group andothers have previously proposed that nonapoptotic CASP3 activityisolated to the uterine myocyte has the capacity to act in a tocolyticfashion through targeting components of the myocyte contractilearchitecture (6, 31, 32). In the present study, we identified the gap

junction protein GJA1 as a previously unidentified target of CASP3and 7 proteolytic action in the pregnant uterine myocyte.Using the hTERT-HM human myometrial cell line, we initially

demonstrated that an ERSR induced chemically by TM couldreadily up-regulate the adaptive prosurvival UPR, as shown in Fig.1, where increased levels of GRP78 allowed the ER to return tohomeostasis. However, prolonged or excessive TM-induced ERSRresulted in elevated DDIT3 levels and, consequently, CASP3 and 7activation and diminished GJA1 levels (Fig. 1A and Fig. S1). In-deed, inhibition of CASP3 and 7 activities allowed GJA1 levels toremain unchanged in the face of an apoptotic challenge (Fig. 2).Several previous studies have identified connexins as sub-

strates of CASP activity and have reported a dramatic decline ingap junction intracellular communication owing to a CASP3-mediated degradation of GJA1 (33). We propose that elevatedCASP3 levels activated by the UPR-ERSR signaling cascadeallow for a decline in uterine GJA1 levels, which suppress thepregnant uterine contractility across gestation. To prove thishypothesis, we induced activation of the UPR-ERSR signalingcascade in the pregnant mouse uterus at E15 by administeringincreasing doses of TM. As shown in Fig. 3, we found elevatedGRP78 levels, indicating an adaptive effort underway to resolvethe TM (0.2 mg/kg)-induced ERSR; however, exposure to aninappropriate/excessive ERSR (TM 1 mg/kg) resulted in an en-hanced UPR that abolished the ERSR, as indicated by decreased

Fig. 5. Subcellular localization of GJA1 and uterine myocyte contractileresponse are disrupted on activation of an excessive ERSR in vitro. (A) Im-munohistochemical analysis of the hTERT-HM cell line identifies GJA1 largelyisolated to the cell membrane in the control and PB (5 mM)- treated uterinemyocytes, which is diminished on exposure to TM (5 μg/mL). Cell membraneGJA1 localization is recovered in the presence of TM when cells are coex-posed to PB. A representative image from three different experiments isshown. (B) Collagen contraction assays reveal reduced contractile re-sponsiveness in the TM-treated hTERT-HM cells, which is reversed on coex-posure of the TM-treated cells to PB. n = 3 per group. Statistical comparisonswere done using one-way ANOVA, followed by the Newman–Keuls multiple-comparison test. *P ≤ 0.05 compared with controls.

Fig. 6. Increased UPR activity modulates pregnant uterine CASP3 and 7 andGJA1 levels in vivo. Treatment of pregnant mice with PB (50 mg/kg) at 1 hbefore and 12 h after TM (1 mg/kg) exposure dampens the consequences ofthe exaggerated TM-induced ERSR, allowing for the recovery of CL CASP3and GJA1 to control levels. n = 6 per treatment group. A representative blotfrom three different experiments is shown. Statistical comparisons weredone using one-way ANOVA, followed by the Newman–Keuls multiple-comparison test. *P ≤ 0.05 compared with controls.

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DDIT3 levels. These events caused a decline in uterine CASP3activation, permitting precocious surges in GJA1 (Fig. 3), ACTA2,and ACTG2 (Fig. S5) levels and the onset of PTB (Table 1).To determine whether we could modulate the PTB phenotype,

we examined the ability of increased chaperone protein action tolimit the TM-induced ERSR in vitro. As shown in Figs. 4 and 5A,administration of PB resolved the TM-induced ERSR, resultingin decreased CASP3 and 7 activation and allowing for increasedGJA1 levels. PB administration also allowed reversal of thetocolytic consequences of ERSR-induced CASP3 and 7 activityas analyzed by 3D collagen contraction analysis (Fig. 5B). Thesedata suggest that, similar to our in vivo analysis shown in Fig. 3,an exaggerated UPR in the context of ERSR promotes anincreasingly contractile phenotype (Fig. 5). In vivo PB administra-tion to pregnant mice at 1 h before and 12 h after TM exposureresolved the ERSR, successfully avoiding the exaggerated UPR and

allowing retention of CASP3, CASP7, GJA1, ACTA2, and ACTG2levels at control levels (Fig. 6 and Fig. S5). It also reversed the TM(0.2 mg/kg)-induced PTB phenotype, allowing live births at term,and delayed the onset of PTB in the 1 mg/kg TM group (Table 2).These data suggest that an ERSR-induced PTB can be pre-

vented by prophylactic up-regulation of the uterine myocyteUPR through preventative administration of a chaperone mimic,such as PB. Interestingly, normal gestational length is main-tained in both the CASP3 and CASP7 null mice (34–36), likelybecause CASP3 and 7 may serve redundant functions for eachother, as has been demonstrated previously in other systems (37,38). Unfortunately, the CASP3 and 7 double-KO mouse is lethal(38), precluding the ability to determine whether maternaluterine CASP3 and 7 activities serve redundant functions in vivowith respect to the maintenance of gestational length.We examined the ability of TM-induced CASP3 and 7 to in-

dependently regulate human uterine myocyte contractility in vitro(Fig. 8). Collagen contraction assays showed that either CASP3 or7, when activated independently, has the capability of maintainingthe uterine myocyte in a quiescent noncontractile phenotype. Incontrast, the elimination of both CASP3 and 7 allowed for in-creased GJA1 levels and contraction rates similar to those ofcontrol untreated cells. These data demonstrate that both CASP3and CASP7 have the capacity to independently maintain uterinequiescence, which likely accounts for the maintenance of gesta-tional length in both the CASP3 null and CASP7 null mice (Fig. 8).Several known modulators of gestational length also have

been shown to modify the UPR-ERSR. Elegant in vitro studies(39) focusing on primary human myometrial cells have demon-strated that when targeted with LPS, the uterine myocyte has thecapacity to induce an exaggerated UPR that is reversed by PB,suggesting that in the uterine myocyte, local inflammation in-deed has the capability of modifying the UPR-ERSR. However,in vivo analysis of the LPS-induced PTB model demonstratedincreased uterine CASP3 activity associated with elevated apoptoticindices such as TUNEL activity isolated to the endometrial, de-cidual, and placental compartments. Modulation of myometrialnonapoptotic CASP3 activity or apoptotic indices in the myometrialcompartment were not affected by LPS administration (40–42). It isclear that progesterone (P4) and progesterone receptor (PR) actionare critical to the maintenance of uterine quiescence (26, 43–46).There is also evidence for the action of P4 and the PR in up-

regulating the ERSR (14, 15). Indeed, pregnant mice treated withP4 2 mg/d from E13 to term demonstrated increased DDIT3 levelsacross gestation, resulting in increased CASP3 activation and re-duced GJA1 levels (Fig. 7A). In contrast, administration of the PRantagonist RU-486 at E13 resulted in rapidly diminished DDIT3levels, causing reduced CASP3 activation and a precocious surgein GJA1 levels (Fig. 7B). Furthermore, the human myometrium

Table 2. Pretreatment with PB rescues the PTB phenotype inTM-treated mice

TM, mg/kg Onset of delivery

0 E19 (3 of 3)0.2 TM E17 (3 of 6)1.0 TM E16 (6 of 6)50 PB E19 (6 of 6)0.2 TM + 50 PB E19 (6 of 6)1.0 TM + 50 PB E18 (6 of 6)

Treatment of pregnant mice with PB (50 mg/kg) at 1 h before and 12 hafter TM (0.2 and 1 mg/kg) exposure rescues the preterm birth phenotype inpregnant mice exposed to TM at 0.2 mg/kg and delayed the onset of PTB inpregnant mice exposed to TM at 1.0 mg/kg by 18 ± 6 h. n = 6 per group.

Fig. 7. Circulating P4 levels and uterine PR action regulate the uterine UPR-ERSR in vivo. (A) Pregnant mice exposed to 2 mg P4/day from E13 to E19display decreased GRP78 and increased DDIT3 levels, resulting in elevated CLCASP3 levels and diminished GJA1 levels. (B) In contrast, uteri isolated frompregnant mice exposed to 150 μg RU486 at E13 exhibit a surge in GRP78 anddramatically diminished DDIT3 levels, resulting in the ablation of CL CASP3,allowing for a precocious elevation in GJA1 levels. n = 6 per treatmentgroup. A representative blot from three different experiments is shown.

Fig. 8. CASP3 and CASP7 act in a compensatory manner. (A) TM independentlyincreases CASP3 and CASP7 activation in hTERT-HM cells transfected with CASP7and CASP3 siRNA respectively. (B) Independent activation of uterine CASP3 orCASP7 causes myocytes to adopt a relaxed phenotype, whereas a decline in thelevels of both active CASP3 and CASP7 increases myocyte contractility. Statisticalcomparisons were done using one-way ANOVA, followed by the Newman–Keulsmultiple-comparison test. *P ≤ 0.05 compared with controls.

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displayed a similar gestational profile as the pregnant mouse, withan elevated UPR associated with later gestational time points (Fig.S8). A recent study demonstrated that laboring human myometrialsamples also exhibit an elevated UPR in both term and pretermlabor (39).In conclusion, we hypothesize that we have uncovered a pre-

viously undefined and physiologically relevant area of pregnantuterine biology that may play a critical role in the regulation ofuterine quiescence and thus gestational length. Our preliminarydata support the hypothesis that transitional events that everypregnancy should accommodate are used to promote a quiescentuterine phenotype across gestation through activation of theERSR process.The ERSR disables the ability of the uterine myocyte to

contract effectively. A uterus that lacks the capacity to withstandthese uterotonic transitional events triggers an inappropriateUPR, which has the capacity to limit the ERSR locally in thepregnant uterus, permitting the uterine muscle cell to regain itscontractile phenotype prematurely allowing for the onset of PTB.

With the growing recognition of an association between ERSRand the UPR with human disease, novel strategies for drugdiscovery and therapeutic intervention ultimately may be used inpreventing PTB.

Materials and MethodsThe conditions for in vitro TM, PB, and P4 administration and UV treatmentwith and without the caspase inhibitor [Quinolyl-valyl-O-methylaspartyl-(2,6-difluorophenoxy)methyl ketone (QVD-OPH)] of the human uterinemyocyte hTERT-HM cell line are described in detail in SI Materials andMethods. The siRNA and hTERT-HM contraction assays and in vivo admin-istration of TM, PB, P4, and RU-486 in pregnant mice are also outlined inSI Materials and Methods. All animal studies were approved by the Uni-versity of Pittsburgh Institutional Animal Care and Use Committee.

ACKNOWLEDGMENTS. This work was supported by the Eunice KennedyShriver National Institute of Child Health and Human Development (Grant1R01 HD065011), the March of Dimes (Grant 21FY12-152), and the WayneState University Perinatal Initiative.

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