Access Type

Open Access Dissertation

Date of Award

January 2018

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Physiology

First Advisor

Jennifer C. Condon

Abstract

Introduction: In this study, we are testing the overarching hypothesis that preconditioning the myometrial UPR allows for the maintenance of non-apoptotic CASP3 activity and thus sustains uterine quiescence. We have previously demonstrated that the pregnant uterus facilitates uterine quiescence through UPR mediated activation of non-apoptotic CASP3, yet the mechanism in which CASP3 utilizes to avoid its apoptotic cell fate is unresolved. There is a growing body of evidence including our own that demonstrates remote and direct preconditioning with minor stresses propagates cytoprotective mechanisms that allow for the avoidance of apoptotic cell death upon exposure to a subsequent more damaging stress, through modulation of the UPR. In this study we demonstrate endogenous pregnant-dependent stress stimuli experienced across gestation act in a preconditioning-like manner to sustain the tocolytic action of non-apoptotic CASP3 within the pregnant uterus in the presence of ensuing stresses and promote an all-around adaptive environment through paracrine and endocrine propagation of a myometrial stress-derived secretome.

Methods: In vitro preconditioning: utilizing the hTERT-HM cell line, uterine myocytes were preconditioned with a minor UPR stress (0.1μg/ml TM) or vehicle and exposed 48 hrs later to a lethal UPR stress (5μg/ml TM) (n=3). In vivo sub-preconditioning: we generated a sub-preconditioned pregnant mouse model by inhibiting the effect UPR mediated stress across gestation (50mg/kg PBA (i.p, E10-15)) or vehicle. Endogenous preconditioned and sub-preconditioned mice were exposed to a mild exogenous stress at E16. Time of delivery was noted. From both the in vitro and in vivo models apoptotic and inflammatory indices were examined. In vitro secretome analysis: SILAC labeled hTERT-HM cells underwent UPR activation by exposure to TM, 5.0μg/ml, 1hr or vehicle. Additionally, SILAC labeled proteins transmitted from the UPR activated myocyte into the media were analyzed via LC/MS/MS to define the UPR generated secretome. In a separate experiment the conditioned media was incubated with a secondary set of naïve hTERT-HM cells, which were examined for UPR activation 48hrs later.

Results: Preconditioning the hTERT-HM cell activated CASP3 in the absence of apoptotic consequences. Reduced NF-kB activation and TNF alpha secretion were also observed. In vivo, the sub-preconditioned mouse CASP3 activation in the uterine compartment transitioned to apoptotic upon exposure to a mild exogenous stress. Furthermore endometrial apoptotic CASP3-dependent iPLA2 activation, increased NF-kB activation and COX1 expression upregulated prostaglandin synthesis, which resulted in a progesterone withdrawal and subsequently a 57% preterm birth rate in the preconditioned mice in comparison to 14% in the endogenously preconditioned animals. Further activation of the UPR in hTERT cells generates a unique stress-generated secretome made up of roughly 90 bone-fide proteins, which propagate systemic adaptive signaling.

Conclusion: We speculate that women who are unable to host an appropriate preconditioning response to gestational stresses are at a significantly increased risk of undergoing spontaneous preterm.

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