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Phosphorylation of connexin 43 at MAPK, PKC or CK1 sites each distinctly alter the kinetics of epidermal wound repair.

ABSTRACT: The gap junction protein connexin 43 (Cx43) is a key player in wound healing, and inhibitors of Cx43, which speed epidermal wound healing, are currently in clinical trials. Here, we provide direct in vivo evidence that specific phosphorylation events on Cx43 change the physiological response during wound healing. Blocking phosphorylation, through mutation of serine residues in Cx43 at the protein kinase C (PKC) or casein kinase 1 (CK1) sites, significantly slowed the rate of wound closure in vivo and in vitro and resulted in a thicker epidermal layer after reepithelialization. Conversely, preventing Cx43 phosphorylation by mitogen-activated protein kinases (MAPKs) through mutation significantly increased the rate of wound closure in vivo Defects in migration, but not proliferation, in all mutants were partially rescued in vitro by changing serine residues to aspartic or glutamic acid. These data prove that specific Cx43 phosphorylation events play an important role at different stages of wound healing. Thus, a clear physiological understanding of the spatiotemporal regulation of kinase activation and consequent effects on gap junctions could lead to a more targeted approach to modulating Cx43 expression during wound healing.

SUBMITTER: Lastwika KJ

PROVIDER: S-EPMC6765181 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Phosphorylation of connexin 43 at MAPK, PKC or CK1 sites each distinctly alter the kinetics of epidermal wound repair.

Lastwika Kristin J KJ Dunn Clarence A CA Solan Joell L JL Lampe Paul D PD

Journal of cell science 20190923 18

The gap junction protein connexin 43 (Cx43) is a key player in wound healing, and inhibitors of Cx43, which speed epidermal wound healing, are currently in clinical trials. Here, we provide direct <i>in vivo</i> evidence that specific phosphorylation events on Cx43 change the physiological response during wound healing. Blocking phosphorylation, through mutation of serine residues in Cx43 at the protein kinase C (PKC) or casein kinase 1 (CK1) sites, significantly slowed the rate of wound closure ...[more]

PMID: 31427427

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Project description:Connexins (C×s) are a family of transmembrane proteins that form hemichannels and gap junctions (GJs) on the cell membranes, and transfer small signaling molecules between the cytoplasm and extracellular space and between connecting cells, respectively. Among C×s, suppressing C×43 expression or function promotes skin wound closure and granulation tissue formation, and may alleviate scarring, but the mechanisms are not well understood. Oral mucosal gingiva is characterized by faster wound closure and scarless wound healing outcome as compared to skin wounds. Therefore, we hypothesized that C×43 function is down regulated during human gingival wound healing, which in fibroblasts promotes expression of genes conducive for fast and scarless wound healing. Cultured gingival fibroblasts expressed C×43 as their major connexin. Immunostaining of unwounded human gingiva showed that C×43 was abundantly present in the epithelium, and in connective tissue formed large C×43 plaques in fibroblasts. At the early stages of wound healing, C×43 was strongly down regulated in wound epithelial cells and fibroblasts, returning to the level of normal tissue by day 60 post-wounding. Blocking of C×43 function by C×43 mimetic peptide Gap27 suppressed GJ-mediated dye transfer, promoted migration, and caused significant changes in the expression of wound healing-associated genes in gingival fibroblasts. In particular, out of 54 genes analyzed, several MMPs and TGF-β1, involved in regulation of inflammation and extracellular matrix (ECM) turnover, and VEGF-A, involved in angiogenesis, were significantly upregulated while pro-fibrotic ECM molecules, including Collagen type I, and cell contractility-related molecules were significantly down regulated. These responses involved MAPK, GSK3α/β and TGF-β signaling pathways, and AP1 and SP1 transcription factors. Thus, suppressed function of C×43 in fibroblasts promotes their migration, and regulates expression of wound healing-associated genes via AP1, SP1, MAPK, GSK3α/β and TGF-β signaling pathways, and may promote fast and scarless wound healing in human gingiva.

Project description:ObjectivesThe gap junction protein, connexin (Cx), plays an important role in maintaining cellular homeostasis and cell proliferation by allowing communication between adjacent cells. Therefore, this study has examined the effect of epidermal growth factor (EGF) on Cx43 and its relationship to proliferation of mouse embryonic stem cells.Materials and methodsExpressions of Cx43, mitogen-activated protein kinases (MAPKs) and cell cycle regulatory proteins were assessed by Western blot analysis. Cell proliferation was assayed with [(3)H]thymidine incorporation. Intercellular communication level was measured by a scrape loading/dye transfer method.ResultsThe results showed that EGF increased the level of Cx43 phosphorylation in a time- (> or =5 min) and dose- (> or =10 ng/mL) dependent manner. Indeed, EGF-induced increase in phospho-Cx43 level was significantly blocked by either AG 1478 or herbimycin A (tyrosine kinase inhibitors). EGF increased Ca(2+) influx and protein kinase C (PKC) translocation from the cytosolic compartment to the membrane compartment. Moreover, pre-treatment with BAPTA-AM (an intracellular Ca(2+) chelator), EGTA (an extracellular Ca(2+) chelator), bisindolylmaleimide I or staurosporine (PKC inhibitors) inhibited the EGF-induced phosphorylation of Cx43. EGF induced phosphorylation of p38 and p44/42 MAPKs, and this was blocked by SB 203580 (a p38 MAPK inhibitor) and PD 98059 (a p44/42 MAPK inhibitor), respectively. EGF or 18alpha-glycyrrhetinic acid (GA; a gap junction inhibitor) increased expression levels of the protooncogenes (c-fos, c-jun and c-myc), cell cycle regulatory proteins [cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2), CDK4 and p-Rb], [(3)H]thymidine incorporation and cell number, but decreased expression levels of the p21(WAF1/Cip1) and p27(Kip1), CDK inhibitory proteins. Transfection of Cx43 siRNA also increased the level of [(3)H]thymidine incorporation and cell number. EGF, 18alpha-GA or transfection of Cx43 siRNA increased 2-DG uptake and GLUT-1 protein expression.ConclusionsEGF-induced phosphorylation of Cx43, which was mediated by the Ca(2+)/PKC, p44/42 and p38 MAPKs pathways, partially contributed to regulation of mouse embryonic stem cell proliferation.

Dataset Information

Phosphorylation of connexin 43 at MAPK, PKC or CK1 sites each distinctly alter the kinetics of epidermal wound repair.

Publications

Phosphorylation of connexin 43 at MAPK, PKC or CK1 sites each distinctly alter the kinetics of epidermal wound repair.

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