Connexin26 and 43 play a role in regulating pro-inflammatory events in the epidermis

Patricia Martin, Erin O'Shaughnessy, Laura-María García-Vega, Afnan Jan, Christopher Bartholomew

Research output: Contribution to journalArticle

Abstract

Dysregulation of Connexin (Cx) expression and function is associated with a range of chronic inflammatory conditions including psoriasis and non-healing wounds. To mimic a pro-inflammatory environment, HaCaT cells, a model human keratinocyte cell line, were challenged with 10 µg/mL peptidoglycan (PGN) isolated from Staphylococcus aureus for 15 minutes to 24 hours in the presence or absence of connexin blockers and/or following CX26, CX43, PANX1 and TLR2 siRNA knock-down (KD). Expression levels of IL-6, IL-8, CX26, CX43, PANX1, TLR2 and Ki67 were assessed by RT-qPCR, western blot and/or immunocytochemistry. NF-kß was blocked with BAY11-7082, CX-channel function was determined by ATP release assays. ELISA monitored IL6 release following PGN challenge in the presence or absence of siRNA or blockers of connexin 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 mRNA expression levels. CX43 protein levels were reduced following 24 h PGN exposure. PGN-induced CX26 and IL-6 expression were also aborted by TLR2-KD and inhibition of NF-kß. ATP and IL-6 release were stimulated following 15 min and 1-24 h challenge with PGN respectively. Release of both agents was inhibited by co-incubation 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-kß, directly activates CX26 and IL-6 expression. CX43 and CX26 maintain pro-inflammatory signalling by permitting ATP release, however PANX1 does not participate.
Original languageEnglish
Pages (from-to)15594-15606
Number of pages13
JournalJournal of Cellular Physiology
Volume234
Issue number9
Early online date2 Feb 2019
DOIs
Publication statusPublished - Sep 2019

Fingerprint

Peptidoglycan
Connexin 43
Epidermis
Interleukin-6
Connexins
Adenosine Triphosphate
Interleukin-8
Small Interfering RNA
Keratinocytes
Psoriasis
Innate Immunity
Gene expression
Staphylococcus aureus
Assays
Western Blotting
Enzyme-Linked Immunosorbent Assay
Immunohistochemistry
Cells
Gene Expression
Cell Line

Keywords

  • Connexin 26
  • Connexin 43
  • Pannexin 1
  • Peptidoglycan
  • Toll-Like-Repector2
  • NF-kB
  • purinergic signalling
  • epidermis
  • inflammation
  • intercellular signalling

Cite this

Martin, Patricia ; O'Shaughnessy, Erin ; García-Vega, Laura-María ; Jan, Afnan ; Bartholomew, Christopher. / Connexin26 and 43 play a role in regulating pro-inflammatory events in the epidermis. In: Journal of Cellular Physiology. 2019 ; Vol. 234, No. 9. pp. 15594-15606.
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abstract = "Dysregulation of Connexin (Cx) expression and function is associated with a range of chronic inflammatory conditions including psoriasis and non-healing wounds. To mimic a pro-inflammatory environment, HaCaT cells, a model human keratinocyte cell line, were challenged with 10 µg/mL peptidoglycan (PGN) isolated from Staphylococcus aureus for 15 minutes to 24 hours in the presence or absence of connexin blockers and/or following CX26, CX43, PANX1 and TLR2 siRNA knock-down (KD). Expression levels of IL-6, IL-8, CX26, CX43, PANX1, TLR2 and Ki67 were assessed by RT-qPCR, western blot and/or immunocytochemistry. NF-k{\ss} was blocked with BAY11-7082, CX-channel function was determined by ATP release assays. ELISA monitored IL6 release following PGN challenge in the presence or absence of siRNA or blockers of connexin 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 mRNA expression levels. CX43 protein levels were reduced following 24 h PGN exposure. PGN-induced CX26 and IL-6 expression were also aborted by TLR2-KD and inhibition of NF-k{\ss}. ATP and IL-6 release were stimulated following 15 min and 1-24 h challenge with PGN respectively. Release of both agents was inhibited by co-incubation 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-k{\ss}, directly activates CX26 and IL-6 expression. CX43 and CX26 maintain pro-inflammatory signalling by permitting ATP release, however PANX1 does not participate.",
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Connexin26 and 43 play a role in regulating pro-inflammatory events in the epidermis. / Martin, Patricia; O'Shaughnessy, Erin; García-Vega, Laura-María; Jan, Afnan; Bartholomew, Christopher.

In: Journal of Cellular Physiology, Vol. 234, No. 9, 09.2019, p. 15594-15606.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Connexin26 and 43 play a role in regulating pro-inflammatory events in the epidermis

AU - Martin, Patricia

AU - O'Shaughnessy, Erin

AU - García-Vega, Laura-María

AU - Jan, Afnan

AU - Bartholomew, Christopher

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AB - Dysregulation of Connexin (Cx) expression and function is associated with a range of chronic inflammatory conditions including psoriasis and non-healing wounds. To mimic a pro-inflammatory environment, HaCaT cells, a model human keratinocyte cell line, were challenged with 10 µg/mL peptidoglycan (PGN) isolated from Staphylococcus aureus for 15 minutes to 24 hours in the presence or absence of connexin blockers and/or following CX26, CX43, PANX1 and TLR2 siRNA knock-down (KD). Expression levels of IL-6, IL-8, CX26, CX43, PANX1, TLR2 and Ki67 were assessed by RT-qPCR, western blot and/or immunocytochemistry. NF-kß was blocked with BAY11-7082, CX-channel function was determined by ATP release assays. ELISA monitored IL6 release following PGN challenge in the presence or absence of siRNA or blockers of connexin 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 mRNA expression levels. CX43 protein levels were reduced following 24 h PGN exposure. PGN-induced CX26 and IL-6 expression were also aborted by TLR2-KD and inhibition of NF-kß. ATP and IL-6 release were stimulated following 15 min and 1-24 h challenge with PGN respectively. Release of both agents was inhibited by co-incubation 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-kß, directly activates CX26 and IL-6 expression. CX43 and CX26 maintain pro-inflammatory signalling by permitting ATP release, however PANX1 does not participate.

KW - Connexin 26

KW - Connexin 43

KW - Pannexin 1

KW - Peptidoglycan

KW - Toll-Like-Repector2

KW - NF-kB

KW - purinergic signalling

KW - epidermis

KW - inflammation

KW - intercellular signalling

U2 - 10.1002/jcp.28206

DO - 10.1002/jcp.28206

M3 - Article

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JO - Journal of Cellular Physiology

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