Uterine Endoplasmic Reticulum Stress-Unfolded Protein Response Regulation of Gestational Length Is Caspase-3 and -7–Dependent

Uterine endoplasmic reticulum stress-unfolded protein response regulation of gestational length is caspase-3 and -7–dependent

Chandrashekara 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 a The endoplasmic reticulum (ER) is the organelle that facilitates potential regulator of uterine quiescence. We also determined protein folding and transport (9), misfolded protein ubiquitination, that during pregnancy, the functional activation of uterine CASP3 and proteasomal degradation. Functional irregularities at the ER is likely governed by an integrated endoplasmic reticulum stress level cause the accumulation of misfolded proteins, leading to ini- response (ERSR) and is consequently limited by an increased tiation of an ERSR (10). A prolonged and/or excessive ERSR has unfolded protein response (UPR). The present study examined been implicated in potentiating increased CASP3 and 7 activation the functional relevance of uterine UPR-ERSR in maintaining (11). In every pregnancy, the uterus experiences physiological and myometrial quiescence and regulating the timing of parturition. biochemical stimuli that in other biological systems trigger an ERSR, including stretch (12), inflammation (13), hormone fluctu- In vitro analysis of the human uterine myocyte hTERT-HM cell line ations (14, 15), hypoxia (16), hyperplasia (17), hypertrophy (18), revealed that tunicamycin (TM)-induced ERSR modified uterine and demand for metabolic fuels (19). myocyte contractile responsiveness. Accordingly, alteration of We propose that the pregnant uterus also may use the ERSR PHYSIOLOGY in vivo uterine UPR-ERSR using a pregnant mouse model signifi- to activate and harness the tocolytic potential of CASP3 and 7, “ ” cantly modified gestational length. We determined that normal allowing it to retain its quiescent phenotype during these periods gestational activation of the ERSR-induced CASP3 and caspase 7 of adaptation. Furthermore, we propose that an inappropriate (CASP7) maintains uterine quiescence through previously uniden- ERSR or UPR mismanagement may modulate uterine CASP3 tified proteolytic targeting of the gap junction protein, alpha 1 and 7 activity, thereby influencing gestational length. We tested (GJA1); however, surprisingly, TM-induced uterine ERSR triggered this hypothesis by manipulating the uterine ERSR and UPR in an exaggerated UPR that eliminated uterine CASP3 and 7 tocolytic the pregnant mouse and monitoring the levels of active CASP3 action precociously. These events allowed for a premature in- and 7 together with the timing of labor. We found that excessive/ crease in myometrial GJA1 levels, elevated contractile responsive- prolonged potentiation of uterine ERSR fails to maintain uterine ness, and the onset of preterm labor. Importantly, a successful CASP3 and 7 levels, owing to the unexpected triggering of a pre- reversal of the magnified ERSR-induced preterm birth phenotype cociously heightened adaptive UPR, which ultimately leads to the could be achieved by pretreatment with 4-phenylbutrate, a chaper- onset of PTB. Coadministration of 4-phenylbutrate (PB) and one protein mimic. tunicamycin (TM) allows for the maintenance of uterine CASP3 and 7 levels, which reverses and rescues the TM-induced PTB endoplasmic reticulum stress | caspase-3 and -7 | unfolded protein phenotype. We have identified GJA1, known to play an essential response | preterm labor | uterus Significance lthough the rates of preterm birth (PTB) continue to decrease Ain the United States, there has been a steady rise in prevalence Preterm birth is a leading cause of neonatal mortality, with a globally over the past decade (1). Multiple risk factors have been poorly understood etiology. The maintenance of uterine quies- associated with preterm labor (2); however, the events that precede cence across gestation is fundamental for term parturition. At and elicit the signals allowing for the onset of premature uterine the molecular level, an integrated uterine endoplasmic reticulum contractions and labor remain unclear. Thus, PTB continues to stress-unfolded protein response (UPR-ERSR) regulates both pose an acute risk for neurodevelopmental and respiratory com- caspase-3 (CASP3) and caspase-7 (CASP7), preserving myometrial quiescence throughout gestation. Here we show that prolonged plications that adversely effect neonatal health (3, 4). In this study, ERSR diminishes the tocolytic potential of uterine CASP3 and 7, we demonstrate that the pregnant rodent uterus uses an integrated causing an increase in myometrial contractile responsiveness unfolded protein response (UPR)-endoplasmic reticulum stress and onset of preterm labor in pregnant mice. Prophylaxis with response (ERSR) pathway to maintain steady levels of activated 4-phenylbutrate, however, maintains active uterine CASP3 and 7 caspase-3 (CASP3) and caspase-7 (CASP7), which preserve uterine and prolongs gestational length. This study establishes a critical quiescence across gestation. We also demonstrate that an increase role for UPR-ERSR in the regulation of pregnant uterine myocyte in the adaptive UPR limits CASP3 and 7 activation to allow the quiescence. induction of both term and preterm labor mediated through increased levels of gap junction protein, alpha 1 (GJA1). Author contributions: P.J. and J.C.C. designed research; C.K., K.O., R.S.M., P.A., and J.C.C. We have identified that CASP3 and 7 play compensatory roles performed research; S.N.C. and J.C.C. contributed new reagents/analytic tools; S.S.H., P.J., in regulating uterine myocyte quiescence. Previous investigations and J.C.C. analyzed data; and J.C.C. wrote the paper. from our laboratory and others have identified a gestationally The authors declare no conflict of interest. regulated activation of nonapoptotic uterine CASP3 during This article is a PNAS Direct Submission. pregnancy (5–7). Furthermore, we have proposed that activation 1To whom correspondence should be addressed. Email: [email protected]. of CASP3 during pregnancy occurs as a result of gestationally This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. regulated increases in uterine ERSR (8). 1073/pnas.1518309112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1518309112 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 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 a uterine UPR-ERSR; however, TM administration at 0.2 mg/kg sucessfully potentiated the UPR-ERSR, as evidenced by increased GRP78 and DDIT3 levels. Uterine myocyte CASP3 levels remained unchanged, suggesting that augmented chaperone action allowed resolution of the 0.2 mg/kg TM-induced ERSR. In contrast, TM administered at 1 mg/kg demonstrated a divergent UPR- ERSR activation profile from our in vitro findings. An exaggerated increase in GRP78 levels, the consequent elimination of DDIT3, and diminished levels of active CASP3 resulted in a surge in GJA1 levels (Fig. 3B). Other organs, such as the heart (Fig. S2A), did not exhibit modified UPR-ERSR/GJA1 levels in response to i.p. administered TM.

Excessive ERS Induces a PTB Phenotype in Pregnant Mice. We examined the timing of the onset of labor in pregnant mice administered a single i.p injection of TM (0, 0.04, 0.08, 0.12, 0.2, and 1 mg/kg) at E15. Administration at <0.12 mg/kg did not alter gestational Fig. 1. ERSR mediates CASP3 activation and consequent GJA1 decline in the length, and all mice delivered live births at term. Pregnant mice given human 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 by TM 0.12 mg/kg had a higher incidence of PTB with delivery of immunoblotting. GRP78 was up-regulated on TM exposure. Prolonged or nonviable neonates at E16 (TM 1.0 mg/kg) and E17 (TM 0.2 mg/kg) excessive exposure to TM (2.5 μg/mL for 48 h or 5 μg/mL 6–48 h) resulted in (Table 1). No maternal morbidity was observed in this study, and activation of the ERSR indicated by an elevation in DDIT3, cleaved (CL) the increased incidence of PTB occurred independently of a de- CASP3, CL CASP7, and CL PARP. (B) On CASP3 and 7 activation (2.5 μg/mL for cline in circulating P4 levels (Fig. S3). 48 h or 5 μg/mL for 6–48 h), diminished levels of GJA1 are observed. A representative blot from three different experiments is shown. PB Attenuates TM-Induced ERSR in Human Uterine Myocytes in Vitro. We next investigated the ability of the chaperone mimic PB to promote an adaptive UPR response in the context of an excessive role in myometrial gap junction intercellular communication (20– TM-induced ERSR. For this, hTERT-HM cells were treated with 22), integrating the signals for active contraction during labor in the 5 μg/mL TM and 5 mM PB concurrently and/or individually. As pregnant uterus, as a target of uterine CASP3 and 7 activity both shown in Fig. 4, TM has the capacity to increase GRP78 and in vitro and in vivo. DDIT3 levels, increase CASP3 and 7 activation, and decrease GJA1 levels. In contrast, pretreatment with PB resolved the TM- Results induced ERSR, thereby suppressing DDIT3 levels, resulting in TM Induces ER Stress and Activates Apoptotic CASP3 Action in Human diminished uterine myocte CASP3 and 7 activation and allowing Uterine Myocytes in Vitro. We examined the functional relevance of GJA1 to remain at control levels. Quantitative RT-PCR analysis UPR-ERSR modulation of uterine myocyte CASP3 and 7 activity confirmed the TM-induced changes in uterine myocyte GJA1 by exposing the telomerase immortalized human uterine myocyte levels at a posttranscriptional level (Fig. S4). (hTERT-HM) cell line (23) to TM. The induction of UPR-ERSR was measured by the abundance of DNA damage-inducible tran- ERSR Activation of CASP3 Disrupts the Uterine Myocyte Contractile script 3 (DDIT3), cleaved CASP3 and CASP7, poly-ADP ribose Apparatus. Immunohistochemical analysis revealed that GJA1, polymerase (PARP) and the chaperone protein, glucose-regulated normally located in the cell membrane, fails to traffic properly in protein 78 kDa (GRP78). As seen in Fig. 1A, GRP78, which acts as the presence of TM-induced ERSR. In contrast, cotreatment a prosurvival adaptive component of the UPR, was up-regulated at each TM exposure (0.5–5 μg/mL). Because the accumulation of excessive misfolded proteins demands additional GRP78, its limited availability can potentiate the onset of an ERSR-induced apoptotic signaling cascade. Accordingly, prolonged or excessive exposure to TM (2.5 μg/mL for 48 h and 5 μg/mL for 6–48 h) resulted in robust induction of DDIT3, cleaved CASP3 and CASP7 levels, and PARP inactivation.

GJA1 Is a Target Substrate for Uterine Myocyte CASP3 Activity in Vitro. Using the hTERT-HM cell line, we also observed that exposure to TM (2.5 μg/mL for 48 h and 5 μg/mL for 6–48 h) resulted in diminished levels of GJA1 in the human uterine myocyte (Fig. 1B). Using the web servers SitePrediciton and Cascleave, we identified 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, and observed increased levels of active CASP3 and 7, corresponding to decreased levels of GJA1. On CASP3 and 7 inhibition, GJA1 levels remained elevated (Fig. 2). Fig. 2. GJA1 is a substrate for active CASP3 in the human uterine myocyte Excessive ERS Negatively Regulates CASP3 Activation in Pregnant in vitro. Using UV light to activate apoptosis in the hTERT-HM cell line, we Mouse Uterus in Vivo. Using the pregnant mouse model, we injected examined GJA1 levels in the absence or presence of 100 μM QVD-OPH. In- vehicle control or TM (0.04, 0.2, and 1 mg/kg i.p.) at embryonic day (E) hibition of CASP3 and CASP7 activation resulted in sustained GJA1 levels. A 15. Uteri were isolated 24 h after TM administration and representative blot from three individual experiments is shown.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1518309112 Kyathanahalli et al. Downloaded by guest on September 30, 2021 Table 1. Excessive uterine ERSR results in the onset of labor in TM-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 per group.

Pretreatment of Pregnant Mice with PB Rescues the TM-Induced PTB Phenotype. Pregnant mice were pretreated with PB (50 mg/kg) at 1 h before and 12 h after TM (0.2 and 1.0 mg/kg) exposure at E15 and then monitored for timing of labor. Table 2 shows that pretreatment with PB rescued the TM (0.2 mg/kg)-ERSR– induced PTB phenotype. In the animals exposed to higher doses of TM (1 mg/kg), PB administration significantly delayed the onset of preterm labor. Treatment with vehicle and PB alone delivered live pups at term. The PB-mediated rescue of the PTB phenotype occurred independent of changes in uterine histone acety- Fig. 3. Excessive ERS negatively regulates CASP3 activity in the pregnant lation (26), isoprenylation rate (27), and PR levels or ratios (28– mouse uterus in vivo. (A) ER homeostasis was maintained in the murine 30) (Fig. S6).

uterus after i.p. administration of TM 0.04 and 0.2 mg/kg. However, TM at PHYSIOLOGY 1 mg/kg triggered increased GRP78 levels, decreased DDIT3 (Fig. S2B), and The Pregnant Uterine UPR-ERSR Is Regulated in a Progesterone- and diminished active CASP3 levels compared with controls (n = 6 per group). (B) Elevated GJA1 levels are observed in the pregnant mouse uterus at 24 h Progesterone Receptor-Dependent Manner. Pregnant mice exposed after i.p. administration of TM (1 mg/kg). A representative blot from three to elevated levels of P4 (2 mg/d) from E13 to E19 demonstrated a different experiments is shown. Statistical comparisons were done using reduced UPR as term approached, resulting in an elevated and pro- one-way ANOVA, followed by the Newman–Keuls multiple-comparison test. longed uterine ERSR, increased CASP3 activation, and diminished *P ≤ 0.05 compared with controls. GJA1 levels (Fig. 7A). In contrast, treatment with the PR inhibitor RU-486 resulted in a dramatic decline in DDIT3 levels and a precocious surge in GRP78 levels, indicating resolution of with PB and TM allows the resolution of the uterine myocte uterine ERSR, at 4 h after RU-486 administration. Rapid ERSR and CASP3 and 7 activation (Fig. 4), permitting suc- elimination of active CASP3 resulted in a premature surge in cessful trafficking of GJA1 to the cell membrane similar to GJA1 levels (Fig. 7B). Because the hTERT-HM cell line used in A control cells and cells treated with PB alone (Fig. 5 ). this study does not express detectable levels of PR, the action of PR and P4 on regulation of the uterine myocyte ERSR in vitro TM-Induced ERSR Modifies Uterine Myocyte Contractile Ability. To was undetectable (Fig. S7). In human pregnancy, similar to the determine the basal contractile phenotype of the human uterine mouse model, term (39–42 wk gestation; n = 6) nonlaboring myocyte in response to changing ERSR levels, we performed 3D myometrium demonstrated an elevation in the UPR, marked by collagen contraction assays with hTERT-HM cells treated with vehicle, 5 μg/mL TM, and 5 mM PB individually and/or concurrently. As shown in Fig. 5B, control and PB-treated cells contracted at a similar rate; however, in the presence of TM, the uterine myocyte retained a noncontractile phenotype. In contrast, hTERT-HM cells coexposed to PB and TM demonstrated increased contractile responsiveness similar to control levels.

In Vivo Administration of PB Modulates Uterine CASP3 Activation and GJA1 Levels. To modulate the in vivo PTB consequences of the exaggerated ERSR induced by TM, we administered PB (50 mg/kg i.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) were administered i.p. individually and served as controls. As shown in Fig. 6, TM administration in vivo induced an exaggerated UPR, resulting in increased GRP78 levels, causing a decline in CASP3 and 7 activity and a dramatic increase in GJA1 levels. Co- administration of TM and PB promoted the maintenance of CASP3, CASP7, and GJA1 at control levels. We previously identified uterine alpha actin (ACTA2) and gamma actin Fig. 4. TM-induced ERSR can be modulated by increased chaperone action (ACTG2) as targets of CASP3 (6), and in the present study, we in vitro. Administration of PB (5 mM) to the hTERT-HM prevents TM (5 μg/mL)- have confirmed their regulation in an ERSR- dependent manner induced ERSR, as indicated by diminished DDIT3, CL CASP3 and 7, and CL PARP in vivo (Fig. S5). levels, allowing for recovery of GJA1 to control levels. n = 3 per group.

Kyathanahalli et al. PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 junction protein GJA1 as a previously unidentified target of CASP3 and 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 could readily up-regulate the adaptive prosurvival UPR, as shown in Fig. 1, where increased levels of GRP78 allowed the ER to return to homeostasis. However, prolonged or excessive TM-induced ERSR resulted in elevated DDIT3 levels and, consequently, CASP3 and 7 activation and diminished GJA1 levels (Fig. 1A and Fig. S1). In- deed, inhibition of CASP3 and 7 activities allowed GJA1 levels to remain unchanged in the face of an apoptotic challenge (Fig. 2). Several previous studies have identified connexins as substrates of CASP activity and have reported a dramatic decline in gap junction intracellular communication owing to a CASP3- mediated degradation of GJA1 (33). We propose that elevated CASP3 levels activated by the UPR-ERSR signaling cascade allow for a decline in uterine GJA1 levels, which suppress the pregnant uterine contractility across gestation. To prove this hypothesis, we induced activation of the UPR-ERSR signaling cascade in the pregnant mouse uterus at E15 by administering increasing doses of TM. As shown in Fig. 3, we found elevated GRP78 levels, indicating an adaptive effort underway to resolve Fig. 5. Subcellular localization of GJA1 and uterine myocyte contractile the TM (0.2 mg/kg)-induced ERSR; however, exposure to an response are disrupted on activation of an excessive ERSR in vitro. (A)Im- inappropriate/excessive ERSR (TM 1 mg/kg) resulted in an en- munohistochemical analysis of the hTERT-HM cell line identifies GJA1 largely hanced UPR that abolished the ERSR, as indicated by decreased isolated to the cell membrane in the control and PB (5 mM)- treated uterine myocytes, which is diminished on exposure to TM (5 μg/mL). Cell membrane GJA1 localization is recovered in the presence of TM when cells are coexposed to PB. A representative image from three different experiments is shown. (B) Collagen contraction assays reveal reduced contractile responsiveness 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 comparisons were done using one-way ANOVA, followed by the Newman–Keuls multiple- comparison test. *P ≤ 0.05 compared with controls.

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

Uterine CASP3 and 7 Have a Compensatory Role for Each Other With Respect to Uterine Myocyte Contractility. hTERT-HM cells were transfected either separately or together with validated siRNAs for CASP3 and CASP7. Western blot analysis confirmed the knockdown 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 cells transfected with CASP7 siRNA. As expected, TM failed to activate either CASP3 or CASP7 in hTERT-HM cells transfected with both CASP3 and CASP7 siRNAs; however, GJA1 levels were reduced in the presence of CASP3 and/or CASP7 (Fig. 8A). Using the aforementioned siRNA-treated hTERT-HM cells, we performed collagen contraction assays in the absence or presence of TM. As shown in Fig. 8B, the presence of either CASP3 or CASP7 resulted in relaxation of the uterine myocyte collagen lattice. In contrast, ablation of both CASP3 and CASP7 allowed successful contraction of collagen gel lattice similar to that seen in control cells. Discussion The present study demonstrates that the pregnant uterine myocyte uses the UPR-ERSR to maintain uterine quiescence by hijacking the tocolytic action of CASP3 and 7 generated in response to the cellular stresses experienced across gestation. We observed that exaggerated, prolonged, or inappropriate uterine ER stress ulti- Fig. 6. Increased UPR activity modulates pregnant uterine CASP3 and 7 and mately causes an imbalanced ERSR, precipitating CASP3 and 7 GJA1 levels in vivo. Treatment of pregnant mice with PB (50 mg/kg) at 1 h before and 12 h after TM (1 mg/kg) exposure dampens the consequences of modulation and resulting in the onset of PTB. Our group and the exaggerated TM-induced ERSR, allowing for the recovery of CL CASP3 others have previously proposed that nonapoptotic CASP3 activity and GJA1 to control levels. n = 6 per treatment group. A representative blot isolated to the uterine myocyte has the capacity to act in a tocolytic from three different experiments is shown. Statistical comparisons were fashion through targeting components of the myocyte contractile done using one-way ANOVA, followed by the Newman–Keuls multiple- architecture (6, 31, 32). In the present study, we identified the gap comparison test. *P ≤ 0.05 compared with controls.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1518309112 Kyathanahalli et al. Downloaded by guest on September 30, 2021 Table 2. Pretreatment with PB rescues the PTB phenotype in TM-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 h after TM (0.2 and 1 mg/kg) exposure rescues the preterm birth phenotype in pregnant mice exposed to TM at 0.2 mg/kg and delayed the onset of PTB in Fig. 8. CASP3 and CASP7 act in a compensatory manner. (A) TM independently pregnant mice exposed to TM at 1.0 mg/kg by 18 ± 6h.n = 6 per group. increases CASP3 and CASP7 activation in hTERT-HM cells transfected with CASP7 and CASP3 siRNA respectively. (B) Independent activation of uterine CASP3 or CASP7 causes myocytes to adopt a relaxed phenotype, whereas a decline in the DDIT3 levels. These events caused a decline in uterine CASP3 levels of both active CASP3 and CASP7 increases myocyte contractility. Statistical activation, permitting precocious surges in GJA1 (Fig. 3), ACTA2, comparisons were done using one-way ANOVA, followed by the Newman–Keuls and ACTG2 (Fig. S5) levels and the onset of PTB (Table 1). multiple-comparison test. *P ≤ 0.05 compared with controls. To determine whether we could modulate the PTB phenotype, we examined the ability of increased chaperone protein action to limit the TM-induced ERSR in vitro. As shown in Figs. 4 and 5A, allowing retention of CASP3, CASP7, GJA1, ACTA2, and ACTG2 administration of PB resolved the TM-induced ERSR, resulting levels at control levels (Fig. 6 and Fig. S5). It also reversed the TM in decreased CASP3 and 7 activation and allowing for increased (0.2 mg/kg)-induced PTB phenotype, allowing live births at term, GJA1 levels. PB administration also allowed reversal of the and delayed the onset of PTB in the 1 mg/kg TM group (Table 2). tocolytic consequences of ERSR-induced CASP3 and 7 activity These data suggest that an ERSR-induced PTB can be pre- as analyzed by 3D collagen contraction analysis (Fig. 5B). These vented by prophylactic up-regulation of the uterine myocyte

data suggest that, similar to our in vivo analysis shown in Fig. 3, UPR through preventative administration of a chaperone mimic, PHYSIOLOGY an exaggerated UPR in the context of ERSR promotes an such as PB. Interestingly, normal gestational length is main- increasingly contractile phenotype (Fig. 5). In vivo PB administra- tained in both the CASP3 and CASP7 null mice (34–36), likely tion to pregnant mice at 1 h before and 12 h after TM exposure because CASP3 and 7 may serve redundant functions for each resolved the ERSR, successfully avoiding the exaggerated UPR and other, 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 maternal uterine CASP3 and 7 activities serve redundant functions in vivo with respect to the maintenance of gestational length. We examined the ability of TM-induced CASP3 and 7 to independently regulate human uterine myocyte contractility in vitro (Fig. 8). Collagen contraction assays showed that either CASP3 or 7, when activated independently, has the capability of maintaining the uterine myocyte in a quiescent noncontractile phenotype. In contrast, the elimination of both CASP3 and 7 allowed for increased GJA1 levels and contraction rates similar to those of control untreated cells. These data demonstrate that both CASP3 and CASP7 have the capacity to independently maintain uterine quiescence, which likely accounts for the maintenance of gestational 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 demonstrated that when targeted with LPS, the uterine myocyte has the capacity to induce an exaggerated UPR that is reversed by PB, suggesting that in the uterine myocyte, local inflammation indeed has the capability of modifying the UPR-ERSR. However, in vivo analysis of the LPS-induced PTB model demonstrated increased uterine CASP3 activity associated with elevated apoptotic indices such as TUNEL activity isolated to the endometrial, de- cidual, and placental compartments. Modulation of myometrial nonapoptotic CASP3 activity or apoptotic indices in the myometrial compartment were not affected by LPS administration (40–42). It is clear that progesterone (P4) and progesterone receptor (PR) action are critical to the maintenance of uterine quiescence (26, 43–46). There is also evidence for the action of P4 and the PR in up- 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 E19 regulating the ERSR (14, 15). Indeed, pregnant mice treated with display decreased GRP78 and increased DDIT3 levels, resulting in elevated CL P4 2 mg/d from E13 to term demonstrated increased DDIT3 levels CASP3 levels and diminished GJA1 levels. (B) In contrast, uteri isolated from across gestation, resulting in increased CASP3 activation and re- pregnant mice exposed to 150 μg RU486 at E13 exhibit a surge in GRP78 and duced GJA1 levels (Fig. 7A). In contrast, administration of the PR dramatically diminished DDIT3 levels, resulting in the ablation of CL CASP3, antagonist RU-486 at E13 resulted in rapidly diminished DDIT3 allowing for a precocious elevation in GJA1 levels. n = 6 per treatment levels, causing reduced CASP3 activation and a precocious surge group. A representative blot from three different experiments is shown. in GJA1 levels (Fig. 7B). Furthermore, the human myometrium

Kyathanahalli et al. PNAS Early Edition | 5of6 Downloaded by guest on September 30, 2021 displayed a similar gestational profile as the pregnant mouse, with With the growing recognition of an association between ERSR an elevated UPR associated with later gestational time points (Fig. and the UPR with human disease, novel strategies for drug S8). A recent study demonstrated that laboring human myometrial discovery and therapeutic intervention ultimately may be used in samples also exhibit an elevated UPR in both term and preterm preventing PTB. labor (39). In conclusion, we hypothesize that we have uncovered a pre- Materials and Methods viously undefined and physiologically relevant area of pregnant The conditions for in vitro TM, PB, and P4 administration and UV treatment uterine biology that may play a critical role in the regulation of with and without the caspase inhibitor [Quinolyl-valyl-O-methylaspartyl- uterine quiescence and thus gestational length. Our preliminary (2,6-difluorophenoxy)methyl ketone (QVD-OPH)] of the human uterine data support the hypothesis that transitional events that every myocyte hTERT-HM cell line are described in detail in SI Materials and pregnancy should accommodate are used to promote a quiescent Methods. The siRNA and hTERT-HM contraction assays and in vivo admin- uterine phenotype across gestation through activation of the istration of TM, PB, P4, and RU-486 in pregnant mice are also outlined in ERSR process. SI Materials and Methods. All animal studies were approved by the Uni- The ERSR disables the ability of the uterine myocyte to versity of Pittsburgh Institutional Animal Care and Use Committee. contract effectively. A uterus that lacks the capacity to withstand these uterotonic transitional events triggers an inappropriate ACKNOWLEDGMENTS. This work was supported by the Eunice Kennedy UPR, which has the capacity to limit the ERSR locally in the Shriver National Institute of Child Health and Human Development (Grant pregnant uterus, permitting the uterine muscle cell to regain its 1R01 HD065011), the March of Dimes (Grant 21FY12-152), and the Wayne contractile phenotype prematurely allowing for the onset of PTB. State University Perinatal Initiative.

1. Bick D (2012) Born too soon: The global issue of preterm birth. Midwifery 28(4):341–342. 24. Verspurten J, Gevaert K, Declercq W, Vandenabeele P (2009) SitePredicting the 2. Romero R, Dey SK, Fisher SJ (2014) Preterm labor: One syndrome, many causes. cleavage of proteinase substrates. Trends Biochem Sci 34(7):319–323. Science 345(6198):760–765. 25. Wang M, et al. (2014) Cascleave 2.0, a new approach for predicting caspase and 3. Mwaniki MK, Atieno M, Lawn JE, Newton CRJC (2012) Long-term neuro- granzyme cleavage targets. Bioinformatics 30(1):71–80. developmental outcomes after intrauterine and neonatal insults: A systematic review. 26. Condon JC, Jeyasuria P, Faust JM, Wilson JW, Mendelson CR (2003) A decline in the Lancet 379(9814):445–452. levels of progesterone receptor coactivators in the pregnant uterus at term may 4. Fraser J, Walls M, McGuire W (2004) Respiratory complications of preterm birth. BMJ antagonize progesterone receptor function and contribute to the initiation of par- 329(7472):962–965. turition. Proc Natl Acad Sci USA 100(16):9518–9523. 5. Shynlova O, et al. (2006) Myometrial apoptosis: Activation of the caspase cascade in 27. Marshall CJ (1993) Protein prenylation: A mediator of protein–protein interactions. the pregnant rat myometrium at midgestation. Biol Reprod 74(5):839–849. Science 259(5103):1865–1866. 6. Jeyasuria P, Wetzel J, Bradley M, Subedi K, Condon JC (2009) Progesterone-regulated 28. Condon JC, Hardy DB, Kovaric K, Mendelson CR (2006) Up-regulation of the pro- caspase 3 action in the mouse may play a role in uterine quiescence during pregnancy gesterone receptor (PR)-C isoform in laboring myometrium by activation of nuclear through fragmentation of uterine myocyte contractile proteins. Biol Reprod 80(5): factor-kappaB may contribute to the onset of labor through inhibition of PR function. 928–934. Mol Endocrinol 20(4):764–775. 7. Stephenson-Famy A, et al. (2012) Antiapoptotic signaling via MCL1 confers resistance 29. Merlino AA, et al. (2007) Nuclear progesterone receptors in the human pregnancy to caspase-3–mediated apoptotic cell death in the pregnant human uterine myocyte. myometrium: evidence that parturition involves functional progesterone withdrawal Mol Endocrinol 26(2):320–330. mediated by increased expression of progesterone receptor-A. J Clin Endocrinol 8. Suresh A, Subedi K, Kyathanahalli C, Jeyasuria P, Condon JC (2013) Uterine endo- Metab 92(5):1927–1933. plasmic reticulum stress and its unfolded protein response may regulate caspase 3 30. Pieber D, Allport VC, Hills F, Johnson M, Bennett PR (2001) Interactions between activation in the pregnant mouse uterus. PLoS One 8(9):e75152. progesterone receptor isoforms in myometrial cells in human labour. Mol Hum 9. Csala M, Kereszturi É, Mandl J, Bánhegyi G (2012) The endoplasmic reticulum as the Reprod 7(9):875–879. extracellular space inside the cell: Role in protein folding and glycosylation. Antioxid 31. Laugwitz KL, et al. (2001) Blocking caspase-activated apoptosis improves contractility Redox Signal 16(10):1100–1108. in failing myocardium. Hum Gene Ther 12(17):2051–2063. 10. Kaufman RJ (1999) Stress signaling from the lumen of the endoplasmic reticulum: 32. Morissette MR, Rosenzweig A (2005) Targeting survival signaling in heart failure. Curr Coordination of gene transcriptional and translational controls. Genes Dev 13(10): Opin Pharmacol 5(2):165–170. 1211–1233. 33. Decrock E, et al. (2009) Connexin-related signaling in cell death: To live or let die? Cell 11. Lin JH, Walter P, Yen TSB (2008) Endoplasmic reticulum stress in disease pathogenesis. Death Differ 16(4):524–536. Annu Rev Pathol 3:399–425. 34. Kuida K, et al. (1996) Decreased apoptosis in the brain and premature lethality in 12. Minamino T, Komuro I, Kitakaze M (2010) Endoplasmic reticulum stress as a thera- CPP32-deficient mice. Nature 384(6607):368–372. peutic target in cardiovascular disease. Circ Res 107(9):1071–1082. 35. Houde C, et al. (2004) Caspase-7 expanded function and intrinsic expression level 13. Burton GJ, Yung HW, Cindrova-Davies T, Charnock-Jones DS (2009) Placental endo- underlies strain-specific brain phenotype of caspase-3–null mice. J Neurosci 24(44): plasmic reticulum stress and oxidative stress in the pathophysiology of unexplained 9977–9984. intrauterine growth restriction and early-onset preeclampsia. Placenta 30(Suppl A): 36. Leonard JR, Klocke BJ, D’Sa C, Flavell RA, Roth KA (2002) Strain-dependent neuro- S43–S48. developmental abnormalities in caspase-3–deficient mice. J Neuropathol Exp Neurol 14. Das SK, et al. (2000) Estrogen targets genes involved in protein processing, calcium 61(8):673–677. homeostasis, and Wnt signaling in the mouse uterus independent of estrogen receptor- 37. Zheng TS, et al. (2000) Deficiency in caspase-9 or caspase-3 induces compensatory alpha and -beta. J Biol Chem 275(37):28834–28842. caspase activation. Nat Med 6(11):1241–1247. 15. Wu ZY, Yu DJ, Soong TW, Dawe GS, Bian JS (2011) Progesterone impairs human ether- 38. Lakhani SA, et al. (2006) Caspases 3 and 7: Key mediators of mitochondrial events of a-go-go-related gene (HERG) trafficking by disruption of intracellular cholesterol apoptosis. Science 311(5762):847–851. homeostasis. J Biol Chem 286(25):22186–22194. 39. Liong S, Lappas M (2014) Endoplasmic reticulum stress is increased after spontaneous 16. Yung HW, Cox M, Tissot van Patot M, Burton GJ (2012) Evidence of endoplasmic re- labor in human fetal membranes and myometrium where it regulates the expression ticulum stress and protein synthesis inhibition in the placenta of non-native women of prolabor mediators. Biol Reprod 91(3):70. at high altitude. FASEB J 26(5):1970–1981. 40. Equils O, et al. (2009) Pretreatment with pancaspase inhibitor (Z-VAD-FMK) delays but 17. Oyadomari S, Araki E, Mori M (2002) Endoplasmic reticulum stress-mediated apo- does not prevent intraperitoneal heat-killed group B Streptococcus-induced preterm ptosis in pancreatic beta-cells. Apoptosis 7(4):335–345. delivery in a pregnant mouse model. Infect Dis Obstet Gynecol 2009:749432. 18. Okada K, et al. (2004) Prolonged endoplasmic reticulum stress in hypertrophic and 41. Savion S, et al. (2002) Apoptosis in the uterus of mice with pregnancy loss. Am J failing heart after aortic constriction: Possible contribution of endoplasmic reticulum Reprod Immunol 47(2):118–127. stress to cardiac myocyte apoptosis. Circulation 110(6):705–712. 42. Li Y, Shibata Y, Zhang L, Kuboyama N, Abiko Y (2011) Periodontal pathogen Ag- 19. Xu C, Bailly-Maitre B, Reed JC (2005) Endoplasmic reticulum stress: Cell life and death gregatibacter actinomycetemcomitans LPS induces mitochondria-dependent apo- decisions. J Clin Invest 115(10):2656–2664. ptosis in human placental trophoblasts. Placenta 32(1):11–19. 20. Hendrix EM, Mao SJT, Everson W, Larsen WJ (1992) Myometrial connexin 43 traf- 43. Lye SJ, Porter DG (1978) Demonstration that progesterone “blocks” uterine activity in ficking and gap junction assembly at term and in preterm labor. Mol Reprod Dev the ewe in vivo by a direct action on the myometrium. J Reprod Fertil 52(1):87–94. 33(1):27–38. 44. Dudley DJ, Branch DW, Edwin SS, Mitchell MD (1996) Induction of preterm birth in 21. Tong D, et al. (2009) A dominant loss-of-function GJA1 (Cx43) mutant impairs par- mice by RU486. Biol Reprod 55(5):992–995. turition in the mouse. Biol Reprod 80(6):1099–1106. 45. Mesiano S, Wang Y, Norwitz ER (2011) Progesterone receptors in the human preg- 22. Ratajczak CK, Muglia LJ (2008) Insights into parturition biology from genetically al- nancy uterus: Do they hold the key to birth timing? Reprod Sci 18(1):6–19. tered mice. Pediatr Res 64(6):581–589. 46. Renthal NE, et al. (2010) miR-200 family and targets, ZEB1 and ZEB2, modulate 23. Condon J, et al. (2002) Telomerase immortalization of human myometrial cells. Biol uterine quiescence and contractility during pregnancy and labor. Proc Natl Acad Reprod 67(2):506–514. Sci USA 107(48):20828–20833.

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