**Karl-Philipp Wesslau, Anabel Stein, Michael Kasper and Kathrin Barth \***

Institute of Anatomy, Dresden University of Technology, D-01307 Dresden, Germany; karl-philipp@wesslau.de (K-P.W.); bella.stein@yahoo.de (A.S.); michael.kasper@tu-dresden.de (M.K.)

**\*** Correspondence: kathrin.barth@tu-dresden.de; Tel.: +49-351-458-6076

Received: 29 March 2019; Accepted: 8 May 2019; Published: 9 May 2019

**Abstract:** The alveolar epithelial cells represent an important part of the alveolar barrier, which is maintained by tight junction proteins, particularly JAM-A, occludin, and claudin-18, which regulate paracellular permeability. In this study, we report on a strong increase in epithelial JAM-A expression in P2X7 receptor knockout mice when compared to the wildtype. Precision-cut lung slices of wildtype and knockout lungs and immortal epithelial lung E10 cells were treated with bleomycin, the P2X7 receptor inhibitor oxATP, and the agonist BzATP, respectively, to evaluate early changes in JAM-A expression. Biochemical and immunohistochemical data showed evidence for P2X7 receptor-dependent JAM-A expression in vitro. Inhibition of the P2X7 receptor using oxATP increased JAM-A, whereas activation of the receptor decreased the JAM-A protein level. In order to examine the role of GSK-3β in the expression of JAM-A in alveolar epithelial cells, we used lithium chloride for GSK-3β inhibiting experiments, which showed a modulating effect on bleomycin-induced alterations in JAM-A levels. Our data suggest that an increased constitutive JAM-A protein level in P2X7 receptor knockout mice may have a protective effect against bleomycin-induced lung injury. Bleomycin-treated precision-cut lung slices from P2X7 receptor knockout mice responded with a lower increase in mRNA expression of JAM-A than bleomycin-treated precision-cut lung slices from wildtype mice.

**Keywords:** JAM-A; P2X7 receptor; mouse lung; alveolar epithelium; bleomycin-induced lung injury; GSK-3β

#### **1. Introduction**

Alveolar epithelial cells (AEC) represent the most vulnerable cells of the distal lung parenchyma and consist of flat AECI type I and cuboidal AECII type II cells in most vertebrates, including humans. Under normal physiological conditions, both cell types are involved in gas exchange and fluid homeostasis [1] and participate in alveolar barrier functions and wound repair processes after lung injury [2]. To maintain cellular polarity and barrier functions of AEC, several types of intercellular junctions exist such as tight junctions (TJ), adherens junctions (AJ), gap junctions, and desmosomes. TJ proteins are particularly important in the regulation of the transcellular permeability of AEC.

Besides their completely different morphological appearance, AECI and AECII specifically differ in their protein pattern, which allows, to a certain degree, their distinction from each other [3]. Knockout of AECI-specific proteins lead to the early death of the animals (T1α knockout, [4], or the lungs exhibit a pathologic phenotype (e.g., caveolin-1 [5], RAGE [6], and aquaporin-5 [7] knockouts). One exception is the P2X7 receptor deficient mouse, which does not exhibit a single sign of histomorphological alterations during its lifetime [8]. The P2X7 receptor (P2X7R) is a ligand-gated ion channel activated by extracellular ATP. In the most distal part of lung, P2X7R is selectively present in AECI [9] and in alveolar macrophages [10]. The intracellular pathways activated by the receptor influence pulmonary inflammation (reviewed in Reference [11]) and the P2X7R knockout (P2X7−/−) lung show altered tight junction protein expression [12].

In experimental studies, P2X7−/<sup>−</sup> deficient mice have presented dramatically reduced lung inflammation with reduced fibrosis markers in the bleomycin (BLM) model [13]. Deletion of P2X7R has a protective effect and the receptor is a therapeutic target for the amelioration of hyperoxia-induced lung injury [14]. P2X7−/<sup>−</sup> animals showed no significant effect of LPS on lung function, alveolar collapse, or fiber deposition in lung parenchyma when compared with wildtype (WT) mice [15].

Treatment with BLM, an anti-cancer agent, is often used as an experimental model of lung injury and pulmonary fibrosis. Molecular changes after BLM exposure include genes encoding growth factors, signaling molecules, and structural proteins, for example, caveolin-1 and diverse junctional proteins. BLM causes an increase in reactive oxygen species and thus induces apoptosis in epithelial and other cells of the lung, leading to disruption of the alveolar barrier. Recently, it was shown that bleomycin-induced lung injury was attenuated in P2X7−/<sup>−</sup> mice [13].

The TJ and AJ are collectively referred to as the apical junctional complex (AJC) and constitute apical intercellular contacts. The AJC contains the key transmembrane proteins occludin, the claudin protein family, and junctional adhesion molecules (JAM) localized to the TJ, as well as E-cadherin in the AJ. JAMs are expressed by a variety of different cells, mainly epithelial cells, endothelial cells, and cells of the immune system, e.g., leukocytes. The importance of JAM-A in regulating barrier function is shown for JAM-A in epithelial and endothelial cells where siRNA mediated loss of JAM-A expression results in enhanced permeability, as determined by transepithelial resistance (TER) [16]. A complex series of poorly understood signaling events establish epithelial barrier function culminating in the formation of mature TJs, whereby JAM-A seems to be important in early events required for TJ assembly. Adhesion complexes are not formed at low Ca2<sup>+</sup> concentration in epithelial cells.

In earlier work, we have shown that high intracellular Ca2<sup>+</sup> content through activation of P2X7R after BLM treatment leads to increased protein kinase (PKC)-β1 in alveolar epithelial cells [17]. The comparison of lung tissues from WT and P2X7−/<sup>−</sup> mice revealed decreased protein and mRNA levels of PKC-β1 and calmodulin (CaM). We demonstrated that the inhibition of P2X7R after BLM treatment also leads to decreased CaM and PKC-β1 content. This indicates that in the BLM model, P2X7R is involved in the regulation of intracellular calcium content and that the PKC-β1 acts downstream of the P2X7R. By stimulating and inhibiting various isoforms, the conventional PKCs, including the isoforms α, β1, β2, and γ are activated by calcium and diacylglycerol. Both factors have been described as triggers of TJ dissolution [18]. In addition to modified TJ protein levels in the P2X7−/−, we also found an increased inactivation of the glycogen synthase kinase (GSK)-3β in the P2X7−/<sup>−</sup> compared to the WT mice [12]. In vitro experiments demonstrated that GSK-3β phosphorylation mediated by PKC enhanced GSK-3β activity. It has also been reported that in vitro GSK-3β is inactivated in the same manner by particular forms of PKCs [19]. The physiological importance of GSK-3β activity in the regulation of the normal epithelial barrier was shown by Severson et al. [20], which implicates the active role of GSK-3β in controlling the expression of the AJC proteins occludin, claudin-1, and E-cadherin.

The aim of this study was to investigate the expression of the tight junction molecule JAM-A in WT and P2X7−/<sup>−</sup> mice and to investigate the involvement of GSK-3β, which has previously been shown to be increased in P2X7−/<sup>−</sup> mice [12]. We further studied the influence of BLM on JAM-A in precision-cut lung slices (PCLS) of WT and P2X7−/<sup>−</sup> mice and in immortal AECI-like E10 cells and whether the GSK-3β(Ser9) phosphorylation changed after BLM treatment. The influence of the inhibition of P2X7R under BLM treatment was studied using the P2X7R inhibitor oxATP. We also investigated whether inactivation of P2X7R led to changes in the phosphorylation of GSK-3β at Ser9 and if this subsequently had an impact on JAM-A protein content.

#### **2. Results**
