Unfolded protein response suppression potentiates LPS-induced barrier dysfunction and inflammation in bovine pulmonary artery endothelial cells

ABSTRACT

The development of novel strategies to counteract diseases related to barrier dysfunction is a priority, since sepsis and acute respiratory distress syndrome are still associated with high mortality rates. In the present study, we focus on the effects of the unfolded protein response suppressor (UPR) 4-Phenylbutyrate (4-PBA) in Lipopolysaccharides (LPS)-induced endothelial injury, to investigate the effects of that compound in the corresponding damage. 4-PBA suppressed binding immunoglobulin protein (BiP) – a UPR activation marker – and potentiated LPS – induced signal transducer and activator of transcription 3 (STAT3) and extracellular signal‑regulated protein kinase (ERK) 1/2 activation. In addition to those effects, 4-PBA enhanced paracellular hyperpermeability in inflamed bovine pulmonary endothelial cells, and did not affect cell viability in moderate concentrations. Our observations suggest that UPR suppression due to 4-PBA augments LPS-induced endothelial injury, as well as the corresponding barrier disruption.

KEYWORDS:

• barrier function
• endoplasmic reticulum
• unfolded protein response

Introduction

Organ function relies on blood vessel permeability, which adapts to physiological needs.¹ The vascular bed of pulmonary, coronary, and skeletal muscle is composed of endothelial cells, forming a restrictive barrier, the vascular endothelium. This is a semi-permeable lining of blood vessels, formed to control the extravasation of nutrients, proteins, and electrolytes to the underlying tissues. Its integrity is important for crucial physiological conditions, including tissue-fluid homeostasis, vascular tone, host defense, and angiogenesis.² Endothelial leakage can impair gas exchange; leading to hypoxia, hypercapnia, acute lung injury (ALI), or acute respiratory distress syndrome ARDS.³ Targeted medicine for ARDS does not exist, hence the mortality rates of the affected individuals are unacceptably high; as evident in the recent pandemic.⁴

Endoplasmic reticulum (ER) accommodates a wide range of chaperones and enzymes for protein maturation. Hypoxia, redox changes, increased secretory load, and nutrient deprivation disrupt ER homeostasis, leading to deposition of misfolded or unfolded proteins in the ER lumen.⁵ Those events activate one or more unfolded protein response (UPR) sensors; namely the protein kinase RNA like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1α (IRE1α). In unstressed conditions, those protein-sensors are attached to the binding immunoglobulin protein (BiP). It has been suggested that UPR is involved in endothelial repairing processes,⁶ and may represent a therapeutic target for ARDS.⁷ UPR modulation appears to hold the capacity to affect barrier function,^8,9 but more information on that topic is needed.

The effects of UPR suppressor 4-phenylbutyric acid (4-PBA) in LPS-induced signal transducer and activator of transcription 3 (STAT3), extracellular signal-regulated kinase 2 (ERK2) activation; as well as in the paracellular permeability of lung endothelial cells have not been investigated yet. Lipopolysaccharides activates Toll-like receptor 4 (TLR-4 receptor), to induce inflammation, and has been extensively used to inflict lung injury in vitro, as well as in vivo.^10,11 4-PBA is a chemical chaperone which prevents the aggregation of misfolded proteins and reduces endoplasmic reticulum stress.¹²

Herein, we report for the first time that 4-PBA potentiates LPS-induced endothelial inflammation in bovine pulmonary artery endothelial cells (BPAEC); as well as the corresponding paracellular hyperpermeability. Moderate doses (0.1–20 mM, 24 hours) of that compound did not affect cell viability. Our study contributes in our understanding of the UPR-related actions in the endothelial context, and suggest that targeting UPR might be a promising pharmacological approach to strengthen barrier function. Future in vivo studies will further explore the potential of the aforementioned possibility.

Materials and methods

Reagents

RIPA buffer (AAJ63306-AP), anti-mouse IgG HRP-linked antibody (95017–554), anti-rabbit IgG HRP-linked antibody (95017–556), nitrocellulose membranes (10063–173), and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (BT142015-5 G) were purchased from VWR (Radnor, PA, USA). The BiP (3183S), phospho-STAT3 (9145S), STAT3 (4904S), phospho-p44/42 MAPK (ERK1/2) (9101S), and p44/42 MAPK (ERK1/2) (9102S) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). The β-actin antibody (A5441), lipopolysaccharides (LPS) (L4130), Corning® Transwell® cell culture inserts (CLS3470), 4-phenylbutyric acid (4-PBA) (P21005) and FITC-Dextran (46945) were available from Sigma-Aldrich (St. Louis, MO, USA).

Cell culture

Bovine pulmonary artery endothelial cells (BPAEC) are available from Genlantis (San Diego, CA) and are maintained in DMEM (45000–304), in which we add fetal bovine serum and 1× penicillin/streptomycin. The cells are left to grow at 37°C, and in 5% CO₂-95% air, and all materials used are obtained from VWR (Radnor, PA).⁹

Western blot analysis

The process is based on the separation of proteins according to their molecular weight, which are consequently transferred to a nitrocellulose membrane, and incubated with the appropriate antibodies. To visualize the secondary antibody, we use the ChemiDoc^TM Touch Imaging System from Bio-Rad (Hercules, CA). Details on the procedures have been previously published.^9,13,14

Cell viability measurement

The cells were seeded onto 96-well culture plates in complete growth media and were treated with 4-PBA (0.1–20 mM) for 24 hours (H). The media was replaced with serum-free media containing 5 mg/mL MTT. An incubation of 3.5 H followed. DMSO dissolved the MTT crystals, and 0.25 H later absorbance (570 nm) was measured utilizing the SPECTROstar Nano® Absorbance Plate Reader by BMG LABTECH.

Fluorescein isothiocyanate (FITC)-Dextran permeability assay

BPAEC were treated with vehicle (0.1% DMSO) or 4-PBA (2 mM) prior to exposure to vehicle (PBS) or LPS (10 μg/ml) (2 H); and FITC-dextran was added. 20 minutes after, fluorescence intensity was measured utilizing a plate reader (see previous paragraph), as previously described.⁹

Densitometry and statistical analysis

ImageJ software (National Institute of Health) was the tool to assess band density, and data are expressed as Mean ± SEM (standard error of the mean). Student’s t-test determined significance (P < 0.05). GraphPad Prism (version 5.01) provided statistical analysis, and n is the number of repeats.

Results

4-PBA suppresses BiP expression in BPAEC

Bovine lung cells were treated with vehicle (0.1% DMSO), or 4-PBA (2 mM) for 12 and 24 hours (H). The results demonstrate that 4-PBA significantly inhibits the expression of the UPR marker BiP (Figure 1a).

Figure 1. 4-PBA potentiates LPS-induced endothelial barrier dysfunction.

Western blot analysis of BiP and β-actin (a) after treatment of BPAEC with vehicle (0.1% DMSO) or 4-PBA (2 mM) for 12 hours (H) and 24 H. Western blot analysis of pSTAT3 and STAT3 (c), pERK1/2 and ERK1/2 (d) after treatment of BPAEC with vehicle (0.1% DMSO) or 4-PBA (2 mM) for 24 H and post-treatment with vehicle (PBS), or LPS (10 μg/ml) (2 H). The blots shown are representative of three independent experiments. The signal intensity of the bands was analyzed by densitometry. Protein levels of BiP, pSTAT3, and pERK1/2 were normalized to β-actin, STAT3, and ERK1/2, respectively. *P < 0.05 vs. vehicle (VEH) and ^$P < 0.05 vs. LPS. Mean ± SEM. (b) BPAEC were treated with either VEH (0.1% DMSO) or 4-PBA (0.1, 1, 2, 5, 10, 15, and 20 mM) for 24 H. Cell viability was evaluated by MTT assay. *P < 0.05 vs VEH, n = 4. Mean ± SEM. (e) BPAEC were seeded onto the trans-well inserts of a 24-well culture plate. After 24 H, the cells were pre-treated with VEH (0.1% DMSO) or 4-PBA (2 mM) for 24 H, and then exposed to VEH (PBS) or LPS (10 µg/ml) for 2 H. After that, cells were incubated with FITC-dextran (1 mg/ml). 20 minutes later, 100 μl of basal media was removed, and fluorescence intensity was measured. *P < 0.05 vs vehicle (VEH) and ^$P < 0.05 vs LPS. n = 4. Mean ± SEM.

Effects of 4-PBA on lung endothelial cell viability

The effects of 4-PBA on cell viability were measured utilizing MTT assay. Cells were seeded on a 96-well plate (10,000 cells/well) and were treated with either vehicle (0.1% DMSO) or 4-PBA (0.1–20 mM) for 24 hours (H). BPAEC treatment with 4-PBA at 0.1–5 mΜ did not significantly affect cell viability. However, higher doses (10–20 mM) of that UPR suppressor reduced cell viability (Figure 1b).

4-PBA potentiates LPS-induced STAT3 activation in BPAEC

The bovine cells were treated with vehicle (0.1% DMSO), or 4-PBA (2 mM) for 24 H, and were then exposed to vehicle (PBS) or LPS (10 µg/ml) for 2 H. The LPS-treated cells exerted higher expression of phosphorylated STAT3, compared to the vehicle-treated cells. Pre-treatment with 4-PBA potentiated LPS-induced STAT3 activation (Figure 1c).

Induction of pERK1/2 expression by 4-PBA and LPS in endothelial cells

The BPAEC were treated with vehicle (0.1% DMSO), or 4-PBA (2 mM) for 24 H prior to vehicle (PBS) or LPS (10 µg/ml) exposure (2 H). The cells treated with LPS or 4-PBA showed increased activation of ERK1/2, compared to the vehicle-treated cells. However, pre-treatment with 4-PBA did not significantly increase phospho (p) ERK1/2 expression, as compared to the LPS-treated cells (Figure 1d).

4-PBA enhances LPS-induced induction barrier dysfunction

FITC-dextran assay was used to evaluate paracellular permeability. BPAEC were seeded onto the trans-well inserts (125000 cells/insert) of a 24-well culture plate to form monolayer. Then, the cells were pretreated with vehicle (0.1% DMSO) or 4-PBA (2 mM) (24 H) prior to a 2-hour treatment with vehicle (PBS) or LPS (10 µg/ml). Our observations suggest that LPS increased paracellular permeability and 4-PBA potentiated that effect (Figure 1e).

Discussion

Inflammation has been associated with endothelial permeability, inducing the formation of endothelial gaps.¹⁵ Those gaps can be transient (acute inflammation), or sustained (chronic inflammation). Tumor necrosis factor-α (TNF-α) induces permeability by activating the Ras homolog gene family, member A (RhoA)/Rho-associated protein kinase (ROCK) pathway to dissociate vascular endothelial protein tyrosine phosphatase (VE-PTP) from cadherin 5, type 2 or VE-cadherin, modulating tight junction proteins.¹⁶ Inflammatory mediators such as nuclear factor-kappa B (NF-κB), extracellular signal-regulated kinase 1/2 (ERK1/2), and Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) also contribute in barrier dysregulation.¹⁷ Figure 1c demonstrates that 4-PBA potentiates LPS-induced STAT3 activation, and Figure 1d shows that this UPR suppressor enhanced pERK1/2 phosphorylation; while it did not affect cell viability in moderate concentrations (Figure 1b).

Lung injury, both direct and indirect, inflicts severe respiratory disorders and death.¹⁸ Medical countermeasures to suppress endothelial inflammation are of urgent need, both Heat shock protein 90 (Hsp90) inhibitors¹⁹ and Growth Hormone Releasing Hormone (GHRH) antagonists^20,21 may represent exciting therapeutic possibilities in lung inflammatory disease due to their anti-inflammatory activities.^8,22 There is very little information on the role of UPR activation in barrier integrity. Recent evidence suggest that it is involved in the barrier-protective effects of growth hormone-releasing hormone antagonists (GHRHAnt)^17,23 and heat shock protein 90 (Hsp90) inhibitors²⁴ in the vasculature.²⁵

GHRH regulates GH secretion, but it has also been involved in a variety of physiological processes²⁶. Receptors responding to that hormone are expressed in extra-hypothalamic tissues and endothelial cells,²⁷ including those of the brain barrier.²⁸ GHRH receptors are involved in inflammation, and GHRHAnt exert strong anti-inflammatory and anti-oxidative effects.^27,29,30 Those antagonists can inhibit bleomycin-induced fibrosis³¹ and prevent LPS-induced bronchoalveolar lavage fluid (BALF) protein concentration in vivo, suggesting their protective effects against edema.³² Hsp90 inhibitors exert similar activities, they can activate UPR,^23,24 and counteract the kifunensine (UPR suppressor)-induced lung endothelial dysfunction.^23,33 They act via the suppression of activated Hsp90, a molecular chaperone in charge with protein stability.³⁴ Interestingly, global UPR modulation due to Brefeldin A and Kifunensine affected endothelial cytoskeletal remodeling by cofilin and myosin light chain 2 (MLC2) activation regulation.^35,36 Cofilin is phosphorylated by Rac1 and severs actin filaments, whereas MLC2 activation signals filamentous actin formation.^9,17 Hence, the Rac1/pCofilin axis strengthens barrier function, and RhoA/MLC2 activation induces hyperpermeability responses.³⁷ P53 has been shown to mediate the aforementioned activities,^38,39 and suppresses STAT3.⁴⁰

Endothelial function can be assessed in vitro via measurements of paracellular and transendothelial permeability. Figure 1e suggests that LPS increases paracellular permeability, and that 4-PBA augments LPS-induced vascular leakage as measured with Dextran-FITC. Indeed, this UPR suppressor itself impaired the endothelium barrier, since the bovine pulmonary artery endothelial cells treated with hat compound presented with barrier dysfunction.

ATF6-related studies revealed that this UPR sensor can support the endothelial barrier integrity.^23,36 The ATF6 inducer AA147 counteracted LPS-induced endothelial barrier disruption by suppressing cofilin and MLC2 activation, as well as by reducing VE-cadherin phosphorylation. ATF6 suppression exerted the opposite effects¹³. Furthermore, PERK-knockout mice presented fibrosis,⁴¹ and BiP silencing suppressed NF-κB activation, and augmented inflammation.^18,42

In conclusion, our study suggest that 4-PBA potentiates LPS-triggered pSTAT3 activation, as well as LPS-induced increase in paracellular permeability. Moreover, UPR suppression in BPAEC resulted to ERK1/2 activation, and this UPR suppressor did not affect cell viability at moderate concentrations. The molecular events beyond those events are to be elucidated in future studies.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

Our work is supported by the Institutional Development Award (IDeA) from NIGMS/NIH (3P20GM103424-21).