P53 in Endothelial Function and Unfolded Protein Response Regulation

Abstract

Vascular barrier dysfunction due to endothelial hyperpermeability has been associated with the pathophysiology of sepsis and severe lung injury, which may inflict acute respiratory distress syndrome (ARDS). Our group is focused on mechanisms operating towards the regulation of endothelial permeability, to contribute in the development of efficient and targeted countermeasures against ARDS. Unfortunately, the ARDS-related deaths in the intensive care units have dramatically increased during the COVID-19 era. The findings described herein inform the relevant scientific and medical community on the relation of P53 and stress responses in barrier function.

1 |. Introduction

The endothelial cells form a semipermeable barrier that regulates blood fluidity and controls vessel permeability to regulate the passage of solutes and small molecules [1]. In several states of human disease (e.g. sepsis, acute respiratory distress syndrome (ARDS)) the impaired endothelial function contributes in disease progression due to hyperpermeability [2]. The consequent endothelial leakage, as well as the infiltration of activated leukocytes into the interstitium, results to lung edema and respiratory failure [3, 4].

Pharmacologic interventions aiming to support the impaired vascular monolayer are of great need. The aberrant accumulation of misfolded proteins in the endoplasmic reticulum (ER) lumen is considered a potential cause of human disease, including cardiovascular disease, sepsis, and acute respiratory distress syndrome (ARDS) [4, 5]. ER is a multifunctional organelle where proteins are folded by chaperons (e.g. heat shock protein 90 and 70) to obtain the appropriate three-dimensional conformation.

Perturbation of ER homeostasis due to cellular stress triggers unfolded protein response (UPR), a phylogenetically conserved signaling pathway. The UPR-initiated transcriptional program tries to restore protein folding defects. If those adaptive responses are inadequate to alleviate ER stress, UPR will shift to proapoptotic signaling to eliminate the irreversible impaired cells [5]. UPR involves a complex network of interconnected signaling pathways- inositol requiring enzyme-1α (IRE-1α), activating transcription factor-6 (ATF6), and protein kinase RNA (PKR)-like ER kinase (PERK); and has now become an attractive target for therapeutic development. Accumulating data have suggested that UPR modulation directly affects barrier function [3, 4, 6].

The Penicillium brefeldianum metabolite Brefeldin A (BFA) exhibits a broad spectrum of antibiotic activity. It is a small hydrophobic compound that induces UPR and inhibits intracellular protein transport from ER to the Golgi complex by preventing association with the corresponding membrane [3, 7]. This lactone antibiotic interferes with vesicle formation by inhibiting the GTP-dependent interaction of ADP-ribosylation factor (ARF) with the Golgi membrane, which in turn blocks the interaction of ARF and cytosolic coat proteins (COPI) with the donor membranes [8]. Kifunensine (KIF), an alkaloid originally isolated from the actinobacterium Kitasatosporia kifunense, is a potent inhibitor of the mannosidase I [6, 9]. It has been reported that KIF-a UPR suppressor-compromises endothelial barrier integrity [6].

In our study we investigate the role of UPR manipulation in P53, a protein that was previously shown to protect the microvasculature against inflammatory stimuli [10]. Since UPR induction is associated with endothelial repair [11], P53 may be involved in those phenomena.

2 |. Materials and Methods

2.1 |. Reagents

KIF (IC15995201), BFA (AAJ62340-MA), anti-mouse IgG horseradish peroxidase (HRP)-linked whole antibody from sheep (95017–554), anti-rabbit IgG HRP-linked whole antibody from donkey (95017–556), radioimmunoprecipitation assay (RIPA) buffer (AAJ63306-AP), and nitrocellulose membranes (10063–173) were obtained from VWR (Radnor, PA). BiP (3183), PDI (2446), ERO1-Lα (3264), MDM2 (86934S), pP53^s392 (9281), and pP53^s46 (2521) antibodies were obtained from Cell Signaling Technology (Danvers, MA). The β-actin antibody (A5441) was purchased from Sigma Aldrich (St Louis, MO), and phospho-IRE1-α antibody (Ser724) (16927) was purchased from Thermo Fisher Scientific (Waltham, MA). MDM4 antibody (ab16058) was obtained from Abcam (Waltham, MA).

2.2 |. Cell culture

The bovine pulmonary arterial endothelial cells (BPAEC) (PB30205) were purchased from Genlantis (San Diego, CA) and maintained at 37°C in a humidified atmosphere of 5% CO2/95% air in Dulbecco’s modified Eagle’s MEM (VWRL0101–0500) medium supplemented with 10% FBS (89510–186), 1× penicillin/streptomycin (97063–708). All reagents were purchased from VWR (Radnor, PA).

2.3|. Western blot analysis

Proteins were isolated from the cells using RIPA buffer. An equal amount of protein was separated by sodium dodecyl sulfate (SDS–PAGE) Tris-HCl gel electrophoresis. Wet transfer was used to transfer the proteins onto nitrocellulose membranes. They were incubated for 60min at room temperature in 5% nonfat dry milk, and were consequently exposed overnight (4°C) to appropriate primary antibodies (1:1,000). The signal for the immunoreactive proteins was developed by using the corresponding secondary antibodies (1:2,000); and visualized in a ChemiDoc Touch Imaging System from Bio-Rad (Hercules, CA) using chemiluminescent substrate (Thermo Scientific). The β-actin antibody was used as a loading control unless otherwise stated.

2.4|. Densitometry and statistical analysis

ImageJ software (National Institutes of Health) was used to perform densitometry of immunoblots. All data are expressed as mean values ± SE (standard error of mean). Student’s t test was performed to determine statistically significant differences among groups. A value of P<0.05 was considered significant. GraphPad Prism 5.01 from GraphPad (CA) was used for data analysis. The letter n represents the number of experimental repeats.

3 |. Results and Discussion

In this study we investigated endothelial P53 regulation in the context of UPR. The heat shock protein 90 (HSP90) client protein P53 regulates major metabolic pathways, required for cellular growth and defense [10]. An emerging body of evidence suggests that inflammation engages P53 in a reciprocal manner [12, 13]. In particular, P53-deficient mice were shown to have more severe autoimmune disorders (arthritis and encephalitis) than their wild-type counterparts [14, 15]. In addition, P53 knockout (KO) mice are more susceptible to acute lung injury due to LPS and bleomycin than the wild type counteracts.

Post-translational P53 modifications include phosphorylation, acetylation, methylation, and ubiquitination. The majority of the covalent modifications take place at intrinsically unstructured linear peptide docking motifs, which flank p53 DNA-binding domain. Those motifs are involved in anchoring allosterically activating enzymes to modify p53. E3 ubiquitin ligases ((e.g. Mouse double minute 2 (MDM2), p53-induced RING-H2 protein (PirH2), constitutively photomorphogenic 1 (COP-1), and C terminus of Hsc70-interacting protein (CHIP)) degrade P53 via a ubiquitin-proteasome dependent mechanism [16]. Six important serine residues on P53 (Ser. 6, Ser. 15, Ser. 20, Ser. 37, Ser. 46, and Ser. 392) have been linked to P53 instability. Moreover, lipopolysaccharides (LPS) induces P53 phosphorylation in Ser. 6, Ser. 15, Ser. 33 and Ser. 392 which in turn promotes endothelial inflammation [13]. UPR inducer tunicamycin and BFA are also P53 inducers [17].

BFA prevented the activation of the Phosphoinositide 3-kinase protein (PI3K/Akt), mammalian target of rapamycin (m-TOR), and nuclear factor kappa B (NF-kB) in keratinocytes; and decreased the production of tumor necrosis factor (TNF)-induced interleukin-15 (IL-15) in HeLa cells [7]. This hydrophobic compound, namely BFA, counteracted KIF-induced barrier dysfunction in lung endothelium [3].

In Figure 1 (FIG.1) we demonstrate that BFA in 2 different concentrations (1 μg/ml, 0.5 μg/ml) induces endoplasmic reticulum oxidoreductin-1 α (ERO1-Lα) (FIG.1A) and protein disulfide isomerase (PDI) (FIG.1B)-which are downstream UPR affecters and responsible for the formation, isomerization, and reduction of disulfide bonds in protein synthesis-as well as pIRE1-α (FIG.1C) and binding protein/glucose regulated protein 78 (BiP/Grp78) (FIG1.D). The former protein is a UPR sensor activated upon ER stress increases, which was reduced in a murine model of LPS-induced acute lung injury [7]. BiP is a molecular chaperone essential for proper barrier function, and a marker of UPR activation that maintain ER transmembrane proteins (ATF6, PERK, IRE1- α) in a repressed state under unstressed conditions [18].

Figure 1:

Western blot analysis of (A) ERO1-Lα and β-actin, (B) PDI and β -actin, (C) pIRE-1α and IRE-1α, (D) BiP and β-actin after treatment of BPAEC with vehicle (0.1% DMSO), or BFA (0.5 μg/ml and 1 μg/ml), or KIF (20 μM and 40 μM) for 24 h. The blots shown are representative of three independent experiments. The signal intensity of the bands was analyzed by densitometry. Protein levels of ERO1-Lα, PDI, and BiP were normalized to β-actin, and pIRE1-α were normalized to IRE1-α. *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle (VEH). Means ± SEM.

It has also been reported that small interfering RNA-mediated inactivation of IRE-1α is related with cytosolic efflux of Ca²⁺ and apoptotic cell death. On the other hand, KIF suppressed UPR in both 20 and 40 μΜ, as reflected in the corresponding UPR markers. The experiments were performed in bovine pulmonary artery endothelial cells per standard procedures previously described [4].

The preclinical findings on the UPR-mediated endothelial function are holding the potential to expand our horizons in the development of targeted therapies against barrier dysfunction. Targeted activation of UPR may accelerate the repair of the dysfunctional endothelial cells. P53 is a cell stress sensor, which primarily acts as a tumor suppressor [10, 19]. Those activities are associated with anti-inflammatory responses, which capacitate the barrier enhancing activities of that transcription factor. P53 which can regulate the balance between the Rac1/RhoA pathways, a crucial event in the modulation of endothelial permeability and cytoskeletal integrity. Rac1 deactivates Cofilin, and RhoA activation results to filamentous actin formation. Hence those pathways exert opposing activities in lung cells [3]. UPR affects P53 expression. In FIG.2A–B it is shown that UPR activation due to BFA reduces MDM2 and MDM4. Both regulators are P53 suppressors, previously shown to be affected by LPS [12]. Moreover, KIF exerted the opposite effects in both 20 and 40 μΜ.

Figure 2:

Western blot analysis of (A) MDM4 and β-actin, (B) MDM2 and β -actin, (C) p-P53S46 and β -actin, and (D) p-P53S392 and β-actin after treatment of BPAEC with vehicle (0.1% DMSO), or BFA (0.5 μg/ml and 1 μg/ml), or KIF (20 μM and 40 μM) for 24 h. The blots shown are representative of three independent experiments. The signal intensity of the bands was analyzed by densitometry. Protein levels of MDM4, MDM2, p-P53S46, and p-P53S392 were normalized to β-actin. *P < 0.05, **P < 0.01 vs. vehicle (VEH). Means ± SEM.

LPS phosphorylates P53, a post -translational modification which is known to trigger P53 degradation [20]. In FIG.2C–D it is demonstrated for the first time that UPR activation due to BFA reduces P53 phosphorylation at S46 and S392. On the other hand, KIF increases p53 phosphorylation. Heat shock protein 90 inhibitors are p53 inducers, able to suppress LPS-induced P53 phosphorylation. Interestingly, those antineoplastic agents are known to be barrier enhancers, indeed [13].

4 |. Conclusions:

The previously reported protective effects of BFA against KIF-induced endothelial hyperpermeability may depend on P53 phosphorylation. However, further studies are needed to shed light to this possibility.

Abbreviations:

ARDS

Acute respiratory distress syndrome

TNF

Tumor necrosis factor

IL

Interleukin

LPS

Lipopolysaccharide

ERO1-Lα

ER oxidoreductase 1α

UPR

Unfolded protein response

BFA

Brefeldin A

KIF

Kifunensine

NF-κB

Nuclear factor kappa B

ER

Endoplasmic reticulum

PERK

Protein kinase RNA like ER kinase

ATF6

Activating transcription factor 6

IRE-1α

Inositol-requiring enzyme-1α

PI3K/Akt

Phosphoinositide 3-kinase

mTOR

Mammalian target of rapamycin

BiP

Binding immunoglobulin protein

GRP94

Glucose regulated protein 94

PDI

Protein disulfide isomerase

Arf

ADP-ribosylation factor

HSP90

Heat shock protein 90

Data availability statement:

The data used to support the findings of this study are available from the corresponding author upon reasonable request.