Connexin 26 and 43 play a role in regulating proinflammatory events in the epidermis

Abstract

Dysregulation of Connexin (CX) expression and function is associated with a range of chronic inflammatory conditions including psoriasis and nonhealing wounds. To mimic a proinflammatory environment, HaCaT cells, a model human keratinocyte cell line, were challenged with 10 µg/ml peptidoglycan (PGN) isolated from Staphylococcus aureus for 15 min to 24 hr in the presence or absence of CX blockers and/or following CX26, CX43, PANX1 and TLR2 small interfering RNA (siRNA) knockdown (KD). Expression levels of IL‐6, IL‐8, CX26, CX43, PANX1, TLR2 and Ki67 were assessed by quantitative real‐time polymerase chain reaction, western blot analysis and/or immunocytochemistry. Nuclear factor kappa β (NF‐κβ) was blocked with BAY 11‐7082, CX‐channel function was determined by adenosine 5′‐triphosphate (ATP) release assays. Enzyme‐linked immunosorbent assay monitored IL6 release following PGN challenge in the presence or absence of siRNA or blockers of CX or purinergic signalling. Exposure to PGN induced IL‐6, IL‐8, CX26 and TLR2 gene expression but it did not influence CX43, PANX1 or Ki67 messenger RNA expression levels. CX43 protein levels were reduced following 24 hr PGN exposure. PGN‐induced CX26 and IL‐6 expression were also aborted by TLR2‐KD and inhibition of NF‐κβ. ATP and IL‐6 release were stimulated following 15 min and 1–24 hr challenge with PGN, respectively. Release of both agents was inhibited by coincubation with CX‐channel blockers, CX26‐, CX43‐ and TLR2‐KD. The IL‐6 response was also reduced by purinergic blockers. CX‐signalling plays a role in the innate immune response in the epidermis. PGN is detected by TLR2, which via NF‐κβ, directly activates CX26 and IL‐6 expression. CX43 and CX26 maintain proinflammatory signalling by permitting ATP release, however, PANX1 does not participate.

A heterozygous mutation in GJB2 (Cx26F142L) associated with deafness and recurrent skin rashes results in connexin assembly deficiencies

“…[10,[33][34][35] Overexpression of CX26 (for example in psoriasis) or these leaky KID hemichannels release ATP from the cells feeding into purinergic signalling pathways and associated pro-inflammatory signalling cascades leading to hyperproliferation loss of epidermal integrity and inflammation. [4,21,36] Cx channel blockers or inhibition of the overexpression of CX26…”

“…can reduce these events and may offer potential therapeutic strategies. [21,37] However, the F142L mutation produced channels with F I G U R E 4 CX26 mutations co-immunoprecipitate with Cx43: HeLa43 cells were transfected to express eGFP, CX26WT, F142L, G12R or D66H-GFP and harvested for co-immunoprecipitation analysis. As a control, HeLa Ohio cells were transfected with eGFP.…”

“…[ 10,33‐35] Overexpression of CX26 (for example in psoriasis) or these leaky KID hemichannels release ATP from the cells feeding into purinergic signalling pathways and associated pro‐inflammatory signalling cascades leading to hyperproliferation loss of epidermal integrity and inflammation. [ 4,21,36] Cx channel blockers or inhibition of the overexpression of CX26 can reduce these events and may offer potential therapeutic strategies. [ 21,37] However, the F142L mutation produced channels with restricted permeability, that was further reduced by exposure to the channel inhibitor CBX.…”

“…[ 4,21,36] Cx channel blockers or inhibition of the overexpression of CX26 can reduce these events and may offer potential therapeutic strategies. [ 21,37] However, the F142L mutation produced channels with restricted permeability, that was further reduced by exposure to the channel inhibitor CBX. This suggests that metabolites exchanged via these restricted channels may influence cell viability pathways.…”

“…Primers against CHOP, GADD34 and the reference gene GAPDH were used in TaqMan PCR reactions (Primer design) as previously described. [15,21]…”