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IκB kinase β regulates epithelium migration during corneal wound healing.

Chen L, Meng Q, Kao W, Xia Y - PLoS ONE (2011)

Bottom Line: We generated triple transgenic Krt12(rtTA/rtTAt)/tet-O-Cre/Ikkβ(F/F) (Ikkβ(ΔCE/ΔCE)) mice by crossing the Krt12-rtTA knock-in mice, which express the reverse tetracycline transcription activator in corneal epithelial cells, with the tet-O-Cre and Ikkβ(F/F) mice.Doxycycline-induced IKKβ ablation occurred in corneal epithelial cells of triple transgenic Ikkβ(ΔCE/ΔCE) mice, but loss of IKKβ did not cause ocular abnormalities in fetal development and postnatal maintenance.Instead, loss of IKKβ significantly delayed healing of corneal epithelial debridement without affecting cell proliferation, apoptosis or macrophage infiltration.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Health, University of Cincinnati Medical Center, Cincinnati, Ohio, United States of America.

ABSTRACT
The IKKβ is known to regulate transcription factor NF-κB activation leading to inflammatory responses. Recent gene knockout studies have shown that IKKβ can orchestrate local inflammatory responses and regulate homeostasis of epithelial tissues. To investigate whether IKKβ has an intrinsic role in epithelial cells, we established an in vivo system in the immune privileged corneal epithelium. We generated triple transgenic Krt12(rtTA/rtTAt)/tet-O-Cre/Ikkβ(F/F) (Ikkβ(ΔCE/ΔCE)) mice by crossing the Krt12-rtTA knock-in mice, which express the reverse tetracycline transcription activator in corneal epithelial cells, with the tet-O-Cre and Ikkβ(F/F) mice. Doxycycline-induced IKKβ ablation occurred in corneal epithelial cells of triple transgenic Ikkβ(ΔCE/ΔCE) mice, but loss of IKKβ did not cause ocular abnormalities in fetal development and postnatal maintenance. Instead, loss of IKKβ significantly delayed healing of corneal epithelial debridement without affecting cell proliferation, apoptosis or macrophage infiltration. In vitro studies with human corneal epithelial cells (HCEpi) also showed that IKKβ was required for cytokine-induced cell migration and wound closure but was dispensable for cell proliferation. In both in vivo and in vitro settings, IKKβ was required for optimal activation of NF-κB and p38 signaling in corneal epithelial cells, and p38 activation is likely mediated through formation of an IKKβ-p38 protein complex. Thus, our studies in corneal epithelium reveal a previously un-recognized role for IKKβ in the control of epithelial cell motility and wound healing.

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IKKβ is required for optimal re-epithelialization of corneal wounds in vivo.(A) The IkkβF/F and IkkβΔCE/ΔCE mice and (B) the C57BL/6 adult mice were subjected to corneal epithelial debridement injury, and some mice were treated with TPCA-1 (5 µM) after injury as indicated. The wounded eyes were stained with green fluorescein for photography at 0 and 18 hours and the wound closure rates were calculated by comparing the wound areas at 18 versus 0 hours. The results are mean ± SD from 18 eyes (9 mice) of each genotype in (A), and from 6 eyes with or without TPCA-1 treatment in (B). Statistical analyses were performed and ***: p<0.001 is considered significant.
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pone-0016132-g003: IKKβ is required for optimal re-epithelialization of corneal wounds in vivo.(A) The IkkβF/F and IkkβΔCE/ΔCE mice and (B) the C57BL/6 adult mice were subjected to corneal epithelial debridement injury, and some mice were treated with TPCA-1 (5 µM) after injury as indicated. The wounded eyes were stained with green fluorescein for photography at 0 and 18 hours and the wound closure rates were calculated by comparing the wound areas at 18 versus 0 hours. The results are mean ± SD from 18 eyes (9 mice) of each genotype in (A), and from 6 eyes with or without TPCA-1 treatment in (B). Statistical analyses were performed and ***: p<0.001 is considered significant.

Mentions: To determine whether IKKβ was required for stress response of the corneal epithelial cells, we introduced corneal epithelial debridement injuries to the IkkβF/F and IkkβΔCE/ΔCE mice and examined the healing processes. We found that the IkkβΔCE/ΔCE mice had clearly a larger wound remained than the IkkβF/F mice at 18 hours after injury (Fig. 3A), suggesting that IKKβ was required for optimal re-epithelialization. To confirm the findings made in the IkkβΔCE/ΔCE mice, we examined the corneal epithelial injury in C57BL/6 mice treated with TPCA-1, a chemical inhibitor of IKKβ. Corneal epithelial debridement was generated on C57BL/6 mice, followed by topical application of either vehicle PBS or TPCA-1 at the wounded corneas. By 18 hours after injury, the epithelial wounds were reduced by 90% in the control PBS-treated corneas, similar to that of the IkkβF/F corneas (Figs. 3A and 3B); however, a larger wound was seen in the TPCA-1 treated mice, mimicking that in the IkkβΔCE/ΔCE mice.


IκB kinase β regulates epithelium migration during corneal wound healing.

Chen L, Meng Q, Kao W, Xia Y - PLoS ONE (2011)

IKKβ is required for optimal re-epithelialization of corneal wounds in vivo.(A) The IkkβF/F and IkkβΔCE/ΔCE mice and (B) the C57BL/6 adult mice were subjected to corneal epithelial debridement injury, and some mice were treated with TPCA-1 (5 µM) after injury as indicated. The wounded eyes were stained with green fluorescein for photography at 0 and 18 hours and the wound closure rates were calculated by comparing the wound areas at 18 versus 0 hours. The results are mean ± SD from 18 eyes (9 mice) of each genotype in (A), and from 6 eyes with or without TPCA-1 treatment in (B). Statistical analyses were performed and ***: p<0.001 is considered significant.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3022035&req=5

pone-0016132-g003: IKKβ is required for optimal re-epithelialization of corneal wounds in vivo.(A) The IkkβF/F and IkkβΔCE/ΔCE mice and (B) the C57BL/6 adult mice were subjected to corneal epithelial debridement injury, and some mice were treated with TPCA-1 (5 µM) after injury as indicated. The wounded eyes were stained with green fluorescein for photography at 0 and 18 hours and the wound closure rates were calculated by comparing the wound areas at 18 versus 0 hours. The results are mean ± SD from 18 eyes (9 mice) of each genotype in (A), and from 6 eyes with or without TPCA-1 treatment in (B). Statistical analyses were performed and ***: p<0.001 is considered significant.
Mentions: To determine whether IKKβ was required for stress response of the corneal epithelial cells, we introduced corneal epithelial debridement injuries to the IkkβF/F and IkkβΔCE/ΔCE mice and examined the healing processes. We found that the IkkβΔCE/ΔCE mice had clearly a larger wound remained than the IkkβF/F mice at 18 hours after injury (Fig. 3A), suggesting that IKKβ was required for optimal re-epithelialization. To confirm the findings made in the IkkβΔCE/ΔCE mice, we examined the corneal epithelial injury in C57BL/6 mice treated with TPCA-1, a chemical inhibitor of IKKβ. Corneal epithelial debridement was generated on C57BL/6 mice, followed by topical application of either vehicle PBS or TPCA-1 at the wounded corneas. By 18 hours after injury, the epithelial wounds were reduced by 90% in the control PBS-treated corneas, similar to that of the IkkβF/F corneas (Figs. 3A and 3B); however, a larger wound was seen in the TPCA-1 treated mice, mimicking that in the IkkβΔCE/ΔCE mice.

Bottom Line: We generated triple transgenic Krt12(rtTA/rtTAt)/tet-O-Cre/Ikkβ(F/F) (Ikkβ(ΔCE/ΔCE)) mice by crossing the Krt12-rtTA knock-in mice, which express the reverse tetracycline transcription activator in corneal epithelial cells, with the tet-O-Cre and Ikkβ(F/F) mice.Doxycycline-induced IKKβ ablation occurred in corneal epithelial cells of triple transgenic Ikkβ(ΔCE/ΔCE) mice, but loss of IKKβ did not cause ocular abnormalities in fetal development and postnatal maintenance.Instead, loss of IKKβ significantly delayed healing of corneal epithelial debridement without affecting cell proliferation, apoptosis or macrophage infiltration.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Health, University of Cincinnati Medical Center, Cincinnati, Ohio, United States of America.

ABSTRACT
The IKKβ is known to regulate transcription factor NF-κB activation leading to inflammatory responses. Recent gene knockout studies have shown that IKKβ can orchestrate local inflammatory responses and regulate homeostasis of epithelial tissues. To investigate whether IKKβ has an intrinsic role in epithelial cells, we established an in vivo system in the immune privileged corneal epithelium. We generated triple transgenic Krt12(rtTA/rtTAt)/tet-O-Cre/Ikkβ(F/F) (Ikkβ(ΔCE/ΔCE)) mice by crossing the Krt12-rtTA knock-in mice, which express the reverse tetracycline transcription activator in corneal epithelial cells, with the tet-O-Cre and Ikkβ(F/F) mice. Doxycycline-induced IKKβ ablation occurred in corneal epithelial cells of triple transgenic Ikkβ(ΔCE/ΔCE) mice, but loss of IKKβ did not cause ocular abnormalities in fetal development and postnatal maintenance. Instead, loss of IKKβ significantly delayed healing of corneal epithelial debridement without affecting cell proliferation, apoptosis or macrophage infiltration. In vitro studies with human corneal epithelial cells (HCEpi) also showed that IKKβ was required for cytokine-induced cell migration and wound closure but was dispensable for cell proliferation. In both in vivo and in vitro settings, IKKβ was required for optimal activation of NF-κB and p38 signaling in corneal epithelial cells, and p38 activation is likely mediated through formation of an IKKβ-p38 protein complex. Thus, our studies in corneal epithelium reveal a previously un-recognized role for IKKβ in the control of epithelial cell motility and wound healing.

Show MeSH
Related in: MedlinePlus