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

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.

Nix induced mitochondrial fission, mitophagy, and myocyte insulin resistance are abrogated by PKA phosphorylation

“…Immunoblotting and Immunoprecipitation: Protein samples were extracted using a RIPA lysis buffer containing protease and phosphatase inhibitors (Santa Cruz). Subcellular organelle fractionation was performed using a Mitochondrial Isolation Kit (Qiagen Qproteome #37612) and a Nuclear/Cytosolic Isolation Kit (Pierce #78833) 60 . Protein determination was performed using a Bio-Rad protein assay kit and proteins were separated by reducing SDS-PAGE and transferred to a PVDF membrane 35,36,[60][61][62] .…”

“…Subcellular organelle fractionation was performed using a Mitochondrial Isolation Kit (Qiagen Qproteome #37612) and a Nuclear/Cytosolic Isolation Kit (Pierce #78833) 60 . Protein determination was performed using a Bio-Rad protein assay kit and proteins were separated by reducing SDS-PAGE and transferred to a PVDF membrane 35,36,[60][61][62] . Immunoblotting was carried out using the following antibodies for analysis: Bnip3L/Nix (CST #12396), Myc-Tag (CST #2278), HA-Tag (CST #3724), Phospho-p70 S6 Kinase Thr389 (CST #9205), p70 S6 Kinase (CST #9202), Phospho-IRS-1 Ser1101 (CST #2385), IRS1 (ProteinTech #17509-1-AP), Phospho-DRP1 Ser637 (CST #4867), DRP1-D6C7 (CST #8570), Rheb E1G1R (CST #13879), PLD6 (Invitrogen #PA5-71510), AIF (CST #5318), MEK (CST # 8727), SERCA2 (CST #9580), and Actin (Santa Cruz #sc-1616).…”

“…Fluorescent staining: MitoSOX, MitoTracker Red CMXRos, and Hoechst 33342 staining was described previously 35,36,60,[63][64][65] . Glucose uptake assay was measured as previously described using the fluorescent D-glucose analog 2NBDG (200 μM; Molecular Probes) 35 .…”

“…Glucose uptake assay was measured as previously described using the fluorescent D-glucose analog 2NBDG (200 μM; Molecular Probes) 35 . Microscopy was performed on an Olympus IX70 inverted microscope (Toronto, ON, Canada) with QImaging Retiga SRV Fast 1394 camera (Surrey, BC, Canada) using NIS Elements AR 3.0 software (Nikon Instruments Inc., Melville, NY, USA), or a Zeiss Axiovert 200 inverted microscope fitted with a Calibri 7 LED Light Source and Axiocam 702 mono camera 36,60 . Quantification, scale bars, background subtract, and processing were done on Fiji (ImageJ) and Zen 2.3 Pro software.…”

Misoprostol Attenuates Cardiomyocyte Proliferation in the Neonatal Heart through Bnip3 and Perinuclear Calcium Signaling

“…Myocardin is a transcriptional co-activator involved in the proliferation, differentiation and maturation of cardiomyocytes, and is a direct transcriptional target of MEF2C [13,16,17,19]. Myocardin's ability to regulate cardiomyocyte proliferation is directly linked to the expression of bone morphogenetic protein (BMP) 10, a growth factor associated with embryonic cardiomyocyte expansion [2,[20][21][22].…”