Full length articleExpression and role of gap junction protein connexin43 in immune challenge-induced extracellular ATP release in Japanese flounder (Paralichthys olivaceus)
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.
References (54)
- et al.
Differentiating connexin hemichannels and pannexin channels in cellular ATP release
FEBS Lett.
(2014) - et al.
The cystic fibrosis transmembrane conductance regulator is a dual ATP and chloride channel
J. Biol. Chem.
(1994) - et al.
Molecular characterization of purinergic receptor P2X4 involved in Japanese flounder (Paralichthys olivaceus) innate immune response and its interaction with ATP release channel Pannexin1
Fish Shellfish Immunol.
(2015) - et al.
Identification and characterization of ATP-gated P2X2 receptor gene dominantly expressed in the Japanese flounder (Paralichthys olivaceus) head kidney macrophages
Fish Shellfish Immunol.
(2016) - et al.
Identification and characterization of a novel NOD-like receptor family CARD domain containing 3 gene in response to extracellular ATP stimulation and its role in regulating LPS-induced innate immune response in Japanese flounder (Paralichthys olivaceus) head kidney macrophages
Fish. Shellfish Immunol.
(2016) - et al.
Cloning and characterization of apoptosis-associated speck-like protein containing a CARD domain (ASC) gene from Japanese flounder Paralichthys olivaceus
Fish Shellfish Immunol.
(2016) - et al.
ATP release channel Pannexin1 is a novel immune response gene in Japanese flounder Paralichthys olivaceus
Fish Shellfish Immunol.
(2014) Role of Connexin/Pannexin containing channels in infectious diseases
FEBS Lett.
(2014)- et al.
Identification and characterization of lipopolysaccharide-induced TNF-alpha factor gene from Japanese flounder Paralichthys olivaceus
Veterinary Immunol. Immunopathol.
(2014) - et al.
Identification and expression analysis of nascent polypeptide-associated complex alpha gene in response to immune challenges in Japanese flounder Paralichthys olivaceus
Fish Shellfish Immunol.
(2015)
Identification and characterization of the cDNAs encoding the two subunits of Chinese mitten crab (Eriocheir sinensis) calcineurin: their implications in stress and immune response
Fish Shellfish Immunol.
The gap junction channel protein connexin 43 is covalently modified and regulated by SUMOylation
J. Biol. Chem.
Regulation of connexin expression by transcription factors and epigenetic mechanisms
Biochim. Biophys. Acta (BBA) – Biomembr.
The gap junction protein connexin43 interacts with the second PDZ domain of the zona occludens-1 protein
Curr. Biol.
Connexin43 phosphorylation and cytoprotection in the heart
Biochim. Biophys. Acta (BBA) - Biomembr.
Connexin43 and the brain transcriptome of newborn mice
Genomics
Gap junctions and connexin-mediated communication in the immune system
Biochim. Biophys. Acta (BBA) – Biomembr.
Evidence that the gap junction protein connexin-43 is the ATP-induced pore of mouse macrophages
J. Biol. Chem.
Cell-cell propagation of NF-kappaB transcription factor and MAP kinase activation amplifies innate immunity against bacterial infection
Immunity
Structural basis for the selective permeability of channels made of communicating junction proteins
Biochim. Biophys. acta
Regulation of cellular atp release
Trans. Am. Clin. Climatol. Assoc.
Shaping immune responses through the activation of dendritic cells–P2 receptors
Purinergic Signal.
Autocrine regulation of T-cell activation by ATP release and P2X(7) receptors
FASEB J.
Purinergic signalling in the immune system. A brief update
Purinergic Signal.
Evidence that release of adenosine triphosphate from endothelial cells during increased shear stress is vesicular
J. Cardiovasc. Pharmacol.
Ecto-F1Fo ATP synthase/F1 ATPase: metabolic and immunological functions
Curr. Opin. Lipidol.
The multidrug resistance (mdr1) gene product functions as an ATP channel
Proc. Natl. Acad. Sci. U. S. A.
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Identification and characterization of two novel functional CD39 splice variants that negatively modulate the proinflammatory response in the Japanese flounder Paralichthys olivaceus
2022, AquacultureCitation Excerpt :In this study, we found that all CD39 splice isoforms were initially downregulated but upregulated at the later stage following both LPS and poly(I:C) stimulation in HKMs. Our previous studies also demonstrated that LPS and poly(I:C) inflammatory challenges can induce eATP release from Japanese flounder gill cells and head kidney cells via pannexin1 and/or connexin43/32 channels (Li et al., 2014a; Li et al., 2020a; Li et al., 2016a). The elevated expression of CD39 splice variants may thus facilitate eATP degradation and therefore prevent excessive inflammation caused by the accumulated eATP in the inflammatory sites.
Extracellular ATP-mediated purinergic immune signaling in teleost fish: A review
2021, AquacultureCitation Excerpt :Interestingly, it was found that the Cx43 and Panx1 hemichannels are coexpressed in Japanese flounder head kidney cells and their expression was upregulated by TLR agonists (LPS and poly(I:C)) stimulation and bacterial infection (Li et al., 2014a; Li et al., 2016b), suggesting that TLR activation may lead to these hemichannels opening and releasing ATP from fish immune cells. Indeed, downregulation or overexpression of these genes by using small interfering RNA (siRNA) and plasmid transfection, respectively, clearly demonstrated that these proteins are engaged in immune challenge-induced ATP release in fish (Li et al., 2014a; Li et al., 2016b). However, the contributions of the individual channel to ATP release are likely unequal.
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S. Li is a senior author.