Misoprostol regulates Bnip3 repression and alternative splicing to control cellular calcium homeostasis during hypoxic stress

Jared T Field,William Diehl-Jones,Tammy L Ivanco,Joseph W Gordon,Matthew D Martens,Donald Chapman,Grant M Hatch,Yan Hai,Wajihah Mughal

doi:10.1038/s41420-018-0104-z

Abstract

The cellular response to hypoxia involves the activation of a conserved pathway for gene expression regulated by the transcription factor complex called hypoxia-inducible factor (HIF). This pathway has been implicated in both the adaptive response to hypoxia and in several hypoxic-ischemic-related pathologies. Perinatal hypoxic injury, often associated with prematurity, leads to multi-organ dysfunction resulting in significant morbidity and mortality. Using a rodent model of neonatal hypoxia and several representative cell lines, we observed HIF1α activation and down-stream induction of the cell death gene Bnip3 in brain, large intestine, and heart which was mitigated by administration of the prostaglandin E1 analog misoprostol. Mechanistically, we determined that misoprostol inhibits full-length Bnip3 (Bnip3-FL) expression through PKA-mediated NF-κB (P65) nuclear retention, and the induction of pro-survival splice variants. We observed that the dominant small pro-survival variant of Bnip3 in mouse cells lacks the third exon (Bnip3ΔExon3), whereas human cells produce a pro-survival BNIP3 variant lacking exon 2 (BNIP3ΔExon2). In addition, these small Bnip3 splice variants prevent mitochondrial dysfunction, permeability transition, and necrosis triggered by Bnip3-FL by blocking calcium transfer from the sarco/endoplasmic reticulum to the mitochondria. Furthermore, misoprostol and Bnip3ΔExon3 promote nuclear calcium accumulation, resulting in HDAC5 nuclear export, NFAT activation, and adaptive changes in cell morphology and gene expression. Collectively, our data suggests that misoprostol can mitigate the potential damaging effects of hypoxia on multiple cell types by activating adaptive cell survival pathways through Bnip3 repression and alternative splicing.

Highlights

Hypoxia is a central element in many diseases of prematurity, including hypoxic/ischemic encephalopathy (HIE)[1], necrotizing enterocolitis (NEC)[2], retinopathy of prematurity[3], and persistent pulmonary hypertension of the newborn (PPHN)[4]
Misoprostol inhibits BCL-2/adenovirus E1B kD-interacting protein 3 (Bnip3)-FL expression in vivo Previously, we demonstrated that misoprostol could inhibit cell death in cultured enterocytes exposed to nutrient stress[22]
We provide evidence that activation of prostaglandin signaling through misoprostol treatment induces pro-survival NF-κB gene expression during hypoxia, resulting in reduced expression of the pro-death Bnip3-FL splice variant and increased expression of prosurvival small Bnip[3] variants

Summary

Introduction

Hypoxia is a central element in many diseases of prematurity, including hypoxic/ischemic encephalopathy (HIE)[1], necrotizing enterocolitis (NEC)[2], retinopathy of prematurity[3], and persistent pulmonary hypertension of the newborn (PPHN)[4]. HIF1α has been shown to increase the expression of members of the Bcl-2 gene family, including the BCL-2/. Depending on the cellular context, Bnip[3] has been previously shown to induce macro-autophagy by disrupting the Beclin-1/Bcl-2 complex[11], promote mitochondrial outer membrane permeability (MOMP) leading to apoptosis[12,13], and trigger mitochondrial permeability transitiondependent necrosis by releasing calcium from the endoplasmic reticulum[12,14]. Canonical NF-κB signaling occurs through repression of Inhibitor of κB (IκB) by the IκB kinase (IKK), other signaling pathways have been shown to alter NF-κB transcriptional activity, co-factor interaction, and alter the nuclear-tocytoplasmic shuttling of the p65 subunit[16,17]. In the context of the Bnip[3] promoter, p65 serves to recruit HDAC1 to repress gene expression[15]

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Results

Conclusion