Ceapin-A7 potentiates lipopolysaccharide-induced endothelial injury

Abstract

Barrier dysfunction is the hallmark of severe lung injury, including acute respiratory distress syndrome. Efficient medical countermeasures to counteract endothelial hyperpermeability do not exist, hence the mortality rates of disorders related to barrier abnormalities are unacceptable high. The unfolded protein response is a highly conserved mechanism, which aims to support the cells against endoplasmic reticulum stress, and ATF6 is a protein-sensor which triggers its activation. In the current study we investigate the effects of ATF6 suppression in LPS-induced endothelial inflammation. Our observations suggest that Ceapin-A7, which is an ATF6 suppressor, potentiates LPS-induced STAT3 and JAK2 activation. Hence ATF6 activation may serve as a new therapeutic possibility towards diseases related to barrier dysfunction.

Introduction

Endothelial cells constantly monitor external stimuli, and increase sensitivity towards endogenous factors [1]. Exposure to toxic chemicals and infectious agents impairs lung function and proteostasis, leading to macrophages activation [2]. Those cells, which reside in pulmonary alveoli, are the first line defense against invading microorganisms and enhance cytokine production [3]. Inflammatory stimuli can disrupt lung endothelial and epithelial barriers, promoting vascular leakage [4]. Cell survival and tissue integrity depend on sustained functional protein networks [5].

The endothelium maintains physiological and immunological functions including vascular tone, inflammatory responses, and coagulation/fibrinolysis [6, 7]. This semipermeable barrier regulates tissue inflammatory response by leukocyte extravasation and cytokine transmigration to injury sites. Endothelial structural and functional integrity is compromised due to bacterial (lipopolysaccharides) and viral (COVID-19) infections [8, 9]. Endothelial barrier dysregulation is associated to life-threatening disorders, and it is the hallmark of acute respiratory distress syndrome (ARDS) [10].

Protein folding into a functional three-dimensional structure is crucial for normal physiology. Newly synthesized polypeptides undergo a sequence of modifications, which take place in the endoplasmic reticulum (ER), where peptides interact with molecular chaperones and folding enzymes [11]. ER maintains an oxidizing environment- in contrast to the cytoplasm-enabling the production of disulfide bonds [12]; and mediates biogenesis, folding, trafficking and degradation.

Increased protein folding demand can result to ER stress due to the excess of immature proteins, to induce ER stress [13, 14]. Unfolded protein response (UPR) activation occurs to alleviate ER stress, so to maintain proteostasis [15, 16]. Recent works reveal that the global UPR suppressor Kifunensine (KIF) induces endothelial hyperpermeability [17], while brefeldin A- a UPR inducer-opposes those events [10]. Activating transcription factor-6 (ATF6) is a protein-transcription factor which senses ER stress, and participates in UPR activation [18]. The present work aims to reveal the role of ATF6 in endothelial inflammation triggered by LPS, utilizing Ceapin-A7. This is a commercially available compound designed to suppress ATF6 [19].

Materials and methods

Reagents:

RIPA buffer (AAJ63306-AP), anti-rabbit (95017–556) IgG HRP-linked antibodies, and nitrocellulose membranes (10063–173) are available from VWR (Radnor, PA). Phospho-JAK2 (3776S), JAK2 (3230S), Phospho-STAT3 (9145S), STAT3 (4904S), Phospho-SAPK/JNK (9251S) and SAPK/JNK (9252S) antibodies were purchased from Cell Signaling Technology (Danvers, MA).

Cell Culture:

Bovine pulmonary artery endothelial cells (BPAEC) are available from Genlantis (San Diego, CA). They were cultured in DMEM (VWRL0101–0500), in which we added 10% fetal bovine serum; as well as 1× penicillin/streptomycin to prevent contaminations. Those endothelial cells were maintained and left to grow at controlled conditions, namely at 37°C in a humidified niche of 5% CO₂–95% air. VWR (Radnor, PA) is the supplier of the material described.

Western Blot Analysis:

RIPA buffer was utilized for cell lysis, and same protein quantity was loaded in each well of the gels used for electrophoresis. 12% of sodium dodecyl sulfate (SDS-PAGE) Tris⋅HCl was used to prepare those gels, and after the end of electrophoresis, all proteins were transferred onto nitrocellulose membranes. The non-specific binding sites of those membranes were blocked with 5% nonfat dry milk for 60 minutes, followed by a 16-hour overnight incubation with the necessary primary antibodies. After the end of that procedure, all membranes were washed for 30 minutes, and a 2-hour incubation with the corresponding secondary antibodies enabled signal detection with the Bio-Rad (Hercules, CA) apparatus ChemiDocTM Touch Imaging System.

Cell Proliferation Assay:

Ten thousand cells were placed in each well of 24-well plates. Ceapin-A7 was added at the concentration of 10 μM, and 24 hours after, the BPAEC were exposed to LPS (1 μg/ml) for 60 minutes. A hemocytometer was used for cell counting, and trypan blue was utilized to ensure that only viable cells are counted.

Results

The ATF6 suppressor Ceapin-A7 potentiates LPS-induced STAT3 activation

First, we evaluated the effects of ATF6 suppression in STAT3 activation due to LPS. To do so, BPAEC were exposed for 24 hours to vehicle (0.1% DMSO), or Ceapin-A7 (15 μM). PBS-which was used as vehicle- or LPS at a concentration of 1 μg/mL were added into the wells for 2 hours. The results depicted in Figure 1A indicate that ATF6 suppression potentiates STAT3 phosphorylation due to LPS.

Figure 1:

Ceapin-A7 potentiates LPS-induced inflammation in BPAEC. Western blot analysis of pSTAT3 and STAT3 (A), pJAK2 and JAK2 (B), pJNK and JNK (C). BPAEC were exposed to Ceapin-A7 (15 μM) or vehicle (0.1% DMSO) (24h); and were post-treated with vehicle (PBS) or LPS (1 μg/mL) (2h). The blots shown are representative of four independent experiments. Signal intensity was analyzed by densitometry, and protein levels of pSTAT3, pJAK2, and pJNK were normalized to STAT3, JAK2, and JNK respectively. *P < 0.05 vs. vehicle (VEH) and ^$P < 0.05 vs. LPS. Data are represented as Means ± SEM. (D). Effects of Ceapin-A7 and LPS in cell proliferation. BPAEC were seeded onto 24-well plates (10,000 cells/well) and were pretreated with vehicle (0.1%DMSO) or Ceapin-A7 (15 μM) for 24h, prior to vehicle (PBS) or LPS exposure (1 μg/mL). *P< 0.05 vs. VEH, ^$P < 0.05 vs. LPS n=3. Means ± SEM.

Ceapin-A7 augments LPS-induced JAK2 phosphorylation

BPAEC were exposed to 0.1% DMSO, or the ATF6 inhibitor Ceapin-A7 at a concentration of 15 μM. After 24 hours the endothelial cells were exposed to PBS, which was used as the vehicle, or 1μg/mL of LPS. Two hours after, the cells were lysed and protein expression was analyzed with Western Blot. The data of Figure 1B indicate that LPS or Ceapin-A7 alone can activate pJAK2. Interestingly, as seen in Figure 1B, Ceapin-A7 potentiates LPS-triggered JAK2 activation (Fig. 1B).

Ceapin-A7 potentiates LPS-induced JNK activation

BPAEC were pre-exposed to 0.1%DMSO or the ATF6 suppressor Ceapin-A7 (15 μM) for 24 hours. After that period, a 2-hour treatment with PBS or LPS (1μg/ml) followed. Figure 1C suggests that ATF6 suppression potentiates LPS-induced JNK activation.

Ceapin-A7 and LPS in cell proliferation

BPAEC were exposed to PBS or Ceapin-A7 (15 μM) for the period of 24 hours. Then, the endothelial cells were treated for 2 hours with PBS, which was used as vehicle, or LPS. 1μg/mL of that endotoxin was used. Our results indicate that LPS reduces cell proliferation, and Ceapin-A7 can enhance that effect (Figure 1D).

Discussion

Protein homeostasis dysregulation has been associated with severe illness, such as cystic fibrosis, neurodegeneration and cancer [20–22]; and ATF6 protects against proteostasis defects [23–25]. This stress-sensing transcription factor fosters adaptive UPR responses by inducing ER protein-encoding genes [26, 27]. It can also interact with nuclear respiratory factor 1 (NRF-1) to alter the transcriptional programming [26]. NRF-1 negatively regulates TGF-β1, and it has been shown to be involved bleomycin-induced lung injury and fibrosis [28].

Ceapin-A7 prevents ER stress-dependent Golgi transport and stabilizes ATF6 oligomers inside the ER lumen; while it suppresses immunoglobulin heavy chain binding protein/glucose regulated protein 78 (BiP/Grp78) [29]. In unstressed conditions BiP binds with the luminal domain of UPR sensors, modulates newly synthesized protein translocation and facilitates their folding and oligomerization [30, 31]. In a murine model of I/R damage, overexpression of BiP exerted cardioprotective effects [32]. It has also been suggested that it exerts anti-inflammatory effects by attenuating tumor necrosis factor- α (TNF-α) production [33].

Inflammation and impaired protein homeostasis are interrelated [34]. Airway stress diseases (COPD, emphysema, asthma) and inflammatory insults promote physical, oxidative, and metabolic stress [35, 36]. ATF6 is a transcriptional regulator of ER homeostasis [37]. The luminal domain and the transmembrane anchor are removed by site-1 (S1P) and site-2 (S2P) proteases, to activate ATF6 [38, 39]. Selective activation of ATF6 by AA147 reduces the production of inflammatory cytokines in peripheral macrophages [40, 41]. The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway decreases the expression of GABA_AR α1 in response to seizures [42]. In ATF6 knockout insulinoma cells, the abundance of both c-Jun N-terminal kinases (JNK) and phospho p38 was increased [43].

AA147 ameliorates LPS-induced endothelial barrier dysfunction by inhibiting two key-proteins, responsible for vascular remodeling. Cofilin, which breaks-down actin filaments, and activated myosin light chain 2 (MLC2). The latter protein associates with filamentous actin formation. On the other hand, Ceapin-A7 exerts opposite effects [9]. Our study suggests that Ceapin-A7 promotes LPS-induced phosphorylation of STAT3, JAK2 and JNK pathways. LPS is the outer membrane of Gram-negative bacteria that disrupts vascular barrier function and triggers toll-like receptors-4 (TLR-4), the most critical pattern-recognition receptor [44]. TLR-4 triggers inflammatory processes [45].

TLR4 activation involves TNF receptor associated factor 6 (TRAF6) and TANK‐binding kinase-1 (TBK-1) to phosphorylate STAT3 on serine 727 [46]. Activation of this cytoplasmic protein (STAT3) is associated with inflammatory cytokine secretion (e.g. IL-6 and IL-10) production in the inflamed lungs [47].

It was previously shown that growth hormone-releasing hormone antagonists (GHRHAnt)-which can induce UPR; inhibit the JAK/STAT pathway [48]. GHRH is a hypothalamic hormone that stimulates the pituitary gland to produce growth hormone, and has been involved in tumor promotion. GHRHAnt are anti-cancer and anti-inflammatory compounds [49, 50]. It was shown that can activate UPR, and counteract endothelial hyperpermeability due to UPR suppression [51]. In addition, those antagonists suppress the production of inducible nitric oxide synthase (iNOS), and exert anti-oxidative effects [52]. GHRHAnt reduce actin stress fiber formation, and deactivate cofilin [51]; to enhance barrier function via P53 induction. Heat shock protein 90 (Hsp90) inhibitors, which induce UPR, exert protective activities against LPS-induced barrier dysfunction [53].

Hsp90 assists toward the maturation of several transcription factors, including P53 and E3 ubiquitin ligase [54]. By reducing chaperone activity, Hsp90 inhibitors restore protein homeostasis [55]. Suppression of Hsp90 by specific pharmacological compounds triggers endothelial UPR activation [56, 57]; and Hsp90 inhibitors enhance barrier function [58, 59].

Our current work in conjunction with previous observations supports that ATF6 is involved in vascular barrier regulation. Future studies in endothelial specific ATF6 mutants will substantiate our observations in models of experimental lung injury.