Elsevier

Fish & Shellfish Immunology

Volume 55, August 2016, Pages 348-357
Fish & Shellfish Immunology

Full length article
Expression and role of gap junction protein connexin43 in immune challenge-induced extracellular ATP release in Japanese flounder (Paralichthys olivaceus)

https://doi.org/10.1016/j.fsi.2016.06.014Get rights and content

Highlights

  • Gap junction protein connexin43 gene (PoCx43) was identified from Japanese Flounder Paralichthys olivaceus.

  • PoCx43 was up-regulated by PAMPs and bacterial challenges.

  • Second messenger cyclic AMP plays an important role in regulating PoCx43 gene expression.

  • PoCx43 is an important conduit for immune challenge-induced extracellular ATP release in fish.

Abstract

Connexin43 (Cx43) is the best characterized gap junction protein that allows the direct exchange of signaling molecules during cell-to-cell communications. The immunological functions and ATP permeable properties of Cx43 have been insensitively examined in mammals. The similar biological significance of Cx43 in lower vertebrates, however, is not yet understood. In the present study we identified and characterized a Cx43 ortholog (termed PoCx43) from Japanese flounder (Paralichthys olivaceus) and investigated its role in immune challenge-induced extracellular ATP release. PoCx43 mRNA transcripts are widely distributed in all tested normal tissues and cells with predominant expression in the brain, and are significantly up-regulated by LPS, poly(I:C) and zymosan challenges and Edwardsiella tarda infections as well, suggesting that PoCx43 expression was modulated by the inflammatory stresses. In addition, cyclic AMP (cAMP), an essential second messenger, also plays an important role in regulating PoCx43 gene expression, by which the PoCx43-mediated gap junctional communication may be regulated. Furthermore, overexpression of PoCx43 in Japanese flounder FG-9307 cells significantly potentiates the LPS- and poly(I:C)-induced extracellular ATP release and this enhanced ATP release was attenuated by pre-incubation with Cx43 inhibitor carbenoxolone. In a complementary experiment, down-regulation of PoCx43 endogenous expression in FG-9307 cells with small interfering RNA also significantly reduced the PAMP-induced extracellular ATP release, suggesting that PoCx43 is an important ATP release conduit under the immune challenge conditions. Finally, we showed that extracellular ATP stimulation led to an increased PoCx43 expression which probably provides a feedback mechanism in regulating PoCx43 expression at the transcriptional level. These findings suggest that PoCx43 is an inducible immune response gene and an important conduit for immune challenge-induced extracellular ATP release in fish.

Introduction

Cellular ATP can be released into the extracellular milieu by a number of different cell types under various physiological and pathophysiological conditions. The released ATP functioned as a potent extracellular signaling molecule can modulate a diversity of cell and organ functions via autocrine and/or paracrine signaling cascades [1], [2]. In immune system, extracellular ATP is an important endogenous danger-associated molecular pattern that can activate innate immune immunity in several different types of immune cells such as dendritic cells [3] and T cells through action on the cell surface-expressed P2 receptors [4], [5].

ATP release can occur through lytic and non-lytic pathways. Several cellular mechanisms have been proposed for the non-lytic ATP release, including 1) vesicular exocytosis that usually occurs in synaptic nerve cells [6], 2) plasma or mitochondrial membrane–associated ATP synthase such as F1Fo ATPase [7], 3) ATP-binding cassette transporters including the cystic fibrosis transmembrane conductance regulator [8] and the multidrug resistance gene product mdr (also known as P-glycoprotein) [9], and 4) ion channels such as connexin (Cx) hemichannels, pannexin channels, maxi-anion channels [10], [11], volume-regulated anion channels [12], and P2X7 receptor channels [13]. The mechanisms for non-lytic ATP release in response to inflammatory stress in lower vertebrates, however, remain incompletely understood.

Our previous investigations revealed that multiple ATP-gated P2X receptors are expressed in a variety of Japanese flounder Paralichthys olivaceus immune tissues and cells [14], [15], [16] and extracellular ATP signaling is engaged in NLRC inflammasome-mediated innate immune response in the Japanese flounder [17], [18]. These observations addressed the importance of extracellular ATP-mediated signaling in fish innate immunity. We have also showed that the release of cytoplasmic ATP into the extracellular space was elicited in the Japanese flounder head kidney cells upon immune stimulations by pathogen-associated molecular pattern ligands such as LPS and poly(I:C). Importantly, this extracellular ATP release could be inhibited by carbenoxolone (CBX) [19], a gap junction protein blocker, suggesting that pannexin and/or Cx is involved in this process. We have previously identified that pannexin1 hemichannel is an important candidate for extracellular ATP release in the Japanese flounder through forming a permeable pore to allow ATP across the plasma membrane [19] and now focus on the role of the gap junction Cx proteins. There are more than 20 Cx members that have been identified in mammalian cells [20]. Several Cx members have been reported to form hemichannels capable of releasing ATP and the most abundant species Cx43 in immune cells has been extensively studied in this regard [2]. For example, Cx43-mediated ATP release have been identified in neutrophils [21] and astrocytes [22].

In addition to serve as a conduit for extracellular ATP release, Cx43 also plays important roles in the immune system [23], [24]. Cx43 has been suggested a key role in pathogen invading, survival and replication and therefore has been proposed as a novel therapeutic target to prevent and treat infectious diseases [24]. It has been evidenced that Cx43 hemichannel signaling plays an important role in the initiation of early innate immune responses in endothelial cells [25]. A variety of studies also confirmed that Cx43 is required for effective activation of dendritic cells [26], NK cells [27] and T cells [28]. In addition, Cx43 was discovered to play an important role in neutrophils recruitment [29], macrophage phagocytosis and the regulation of host response to microbial infection [30].

Even through the role of Cx43 for ATP release and its immune significance have been widely recognized in mammals, the similar functions in fish are still lacking. In the present study we isolated a Cx43 cDNA ortholog (termed PoCx43) from Japanese flounder P. olivaceus and revealed that PoCx43 is a highly inducible innate immune response gene. Using combined biochemistry and complementary molecular approaches, we also for the first time show that PoCx43 is an important conduit for the immune challenge-induced extracellular ATP release in fish. These findings will facilitate our understandings for the details of extracellular ATP-mediated innate immune signaling in fish.

Section snippets

Fish maintenance and tissue collection

Japanese flounder P. olivaceus were obtained from a local fish farm in Dagang, Tianjin, China and maintained in an aerated recirculating seawater system in the laboratory at 21 °C for two weeks before experimentations. Fish were clinically examined before experimentation and only healthy animals without any pathological signs were selected for experiments. Individual tissues form Japanese flounders were separated and collected aseptically as described in the previous study [19].

cDNA preparation and amplification of Japanese flounder PoCx43 cDNA

Total RNA from P

Sequence analysis of the Japanese flounder connexin43

The full-length Japanese flounder Cx43 (PoCx43) cDNA is 1325 bp (GenBank accession No. KU886276), which encodes a polypeptide of 388 amino acid residues with a calculated molecular mass of 43.6 kDa and an isoelectric point of 9.1 (Supplementary Fig. 1). Blast against the GenBank database revealed that PoCx43 protein is highly conserved and shares more than 70% and 90% amino acid sequence identities with the available mammalian and teleost Cx43 proteins, respectively. Structurally, the Japanese

Discussion

In this study we characterized the sequence, expression and ATP permeability of PoCx43, the most widely distributed Cx protein family member, from bony fish Japanese flounder P. olivaceus. Cx43 is a highly evolutionary conserved protein. In agreement with this property, the deduced PoCx43 protein shows a high degree of sequence conservation (>70% sequence identities with the currently available vertebrate Cx43 proteins) and its most conserved region is located at the N-terminus. Accordingly,

Acknowledgements

This work was supported by funds from the National Natural Science Foundation of China (No. 31572645), the Science Promotion Program for Young Scholars provided by Tianjin Normal University (No. 52XC1503) and the National Key Technology R&D Program (Nos. 2011BAD13B07 and 2011BAD13B04). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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