Uterine Endoplasmic Reticulum Stress-Unfolded Protein Response Regulation of Gestational Length Is Caspase-3 and -7–Dependent
Total Page：16
File Type：pdf, Size：1020Kb
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 in- creased 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 con- trast, 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.