Abstract
Cytoplasmic membrane-bound connexin 43 (Cx43) proteins oligomerize into hexameric channels (hemichannels) that can sometimes dock with hemichannels on adjacent cells to form gap junctional (GJ) channels. However, the possible role of Cx43 hemichannels in sterile and infectious inflammatory diseases has not been adequately defined due to the lack of selective interventions. Here we report that a proinflammatory mediator, the serum amyloid A (SAA), resembled bacterial endotoxin by stimulating macrophages to up-regulate Cx43 expression and double-stranded RNA-activated protein kinase R (PKR) phosphorylation in a TLR4-dependent fashion. Two well-known Cx43 mimetic peptides, the GAP26 and TAT-GAP19, divergently affected macrophage hemichannel activities in vitro, and differentially altered the outcome of lethal sepsis in vivo. By screening a panel of Cx43 mimetic peptides, we discovered that one cysteine-containing peptide, P5 (ENVCYD), effectively attenuated hemichannel activities, and significantly suppressed endotoxin-induced release of ATP and HMGB1 in vitro. In vivo, the P5 peptide conferred a significant protection against hepatic ischemia/reperfusion injury and lethal microbial infection. Collectively, these findings have suggested a pathogenic role of Cx43 hemichannels in sterile injurious as well as infectious inflammatory diseases possibly through facilitating extracellular ATP efflux to trigger PKR phosphorylation/activation.
Highlights
HMGB1 can be passively released from damaged cells[12] following ischemia/reperfusion (I/R) injury[13], thereby serving as a damage-associated molecular pattern molecule (DAMP)
We reported for the first time that a newly recognized proinflammatory mediator, SAA9, resembled the crude endotoxin, and up-regulated Connexin 43 (Cx43) expression and parallel protein kinase R (PKR) phosphorylation in a TLR4-dependent fashion
The number of transferase dUTP nick-end labeling (TUNEL)-positive green cells was expressed as an average percentage of total DAPI-positive blue cells in 5–8 randomly selected fields. *P < 0.05 versus “Sham” control; #P < 0.05 versus “cecal ligation and puncture (CLP)” group
Summary
HMGB1 can be passively released from damaged cells[12] following ischemia/reperfusion (I/R) injury[13], thereby serving as a damage-associated molecular pattern molecule (DAMP). The cytoplasmic membrane-bound Cx43 oligomerize to form hexameric hemichannels[21], which can dock with the hemichannels on adjacent cells to form GJ channels in non-immune cells (of the heart, brain and vasculature) to facilitate intercellular communications[22,23,24]. It was previously unknown whether Cx43 hemichannel occupies a possible role in the pathogenesis of injury- and infection-elicited excessive inflammation due to lack of selective interventions. We developed a class of novel mimetic peptides (P5) that selectively inhibited macrophage hemichannel activities without impairing GJ communications, and further explored their therapeutic potential in animal models of hepatic I/R injury and lethal sepsis
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