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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 dispensable for the maintenance of mouse corneal epithelium.The adult double (IkkβF/F) and triple (IkkβΔCE/ΔCE) transgenic mice were fed with Dox-chow for 30 days. (A) Genomic DNA was isolated from the corneal epithelial cells and subjected to PCR genotyping using primers specific for the IkkβΔ (deleted allele), IkkβF (floxed allele) and Gapdh. The triple transgenic mice lost IkkβF and acquired IkkβΔ alleles. (B) The eyes were photographed and (C) their tissue sections were subjected to histological analyses by H&E staining and molecular characterization by immunostaining (red) and nuclei were identified by DAPI staining (blue). The TUNEL assay was used to detect apoptosis, BrdU incorporation was used for proliferation, and the expression of KRT12 and KRT14 was used to evaluate corneal (KRT12) and basal (KRT14) epithelial differentiation. The activation of the IKK pathway was evaluated by p65 nuclear translocation and pictures were taken at low and high (rectangle inserts) magnifications. The boundaries of corneal epithelium (epi) and stroma (str) were marked with dotted lines. The picture represents at least 3 slides of each mouse and 2 mice of each genotype used for the studies.
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pone-0016132-g002: IKKβ is dispensable for the maintenance of mouse corneal epithelium.The adult double (IkkβF/F) and triple (IkkβΔCE/ΔCE) transgenic mice were fed with Dox-chow for 30 days. (A) Genomic DNA was isolated from the corneal epithelial cells and subjected to PCR genotyping using primers specific for the IkkβΔ (deleted allele), IkkβF (floxed allele) and Gapdh. The triple transgenic mice lost IkkβF and acquired IkkβΔ alleles. (B) The eyes were photographed and (C) their tissue sections were subjected to histological analyses by H&E staining and molecular characterization by immunostaining (red) and nuclei were identified by DAPI staining (blue). The TUNEL assay was used to detect apoptosis, BrdU incorporation was used for proliferation, and the expression of KRT12 and KRT14 was used to evaluate corneal (KRT12) and basal (KRT14) epithelial differentiation. The activation of the IKK pathway was evaluated by p65 nuclear translocation and pictures were taken at low and high (rectangle inserts) magnifications. The boundaries of corneal epithelium (epi) and stroma (str) were marked with dotted lines. The picture represents at least 3 slides of each mouse and 2 mice of each genotype used for the studies.

Mentions: To evaluate the roles of IKKβ in maintenance of corneal homeostasis, we fed the adult double (Krt12rtTA/rtTA/IkkβF/F) and triple (Krt12rtTA/rtTA/tetO-Cre/IkkβF/F) transgenic mice Dox-chow for 30 days. To confirm the induction of Ikkβ gene deletion, we examined the genomic DNA isolated from corneal epithelial cells. By PCR, we detected only the IkkβF allele in cells isolated from double transgenic mice, whereas, we found only the IkkβΔ allele in cells isolated from triple transgenic mice (Fig. 2A). The triple transgenic mice with induced corneal epithelium-specific Ikkβ ablation are hereafter referred to as IkkβΔCE/ΔCE, whereas the control double transgenic mice are referred to as IkkβF/F.


IκB kinase β regulates epithelium migration during corneal wound healing.

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

IKKβ is dispensable for the maintenance of mouse corneal epithelium.The adult double (IkkβF/F) and triple (IkkβΔCE/ΔCE) transgenic mice were fed with Dox-chow for 30 days. (A) Genomic DNA was isolated from the corneal epithelial cells and subjected to PCR genotyping using primers specific for the IkkβΔ (deleted allele), IkkβF (floxed allele) and Gapdh. The triple transgenic mice lost IkkβF and acquired IkkβΔ alleles. (B) The eyes were photographed and (C) their tissue sections were subjected to histological analyses by H&E staining and molecular characterization by immunostaining (red) and nuclei were identified by DAPI staining (blue). The TUNEL assay was used to detect apoptosis, BrdU incorporation was used for proliferation, and the expression of KRT12 and KRT14 was used to evaluate corneal (KRT12) and basal (KRT14) epithelial differentiation. The activation of the IKK pathway was evaluated by p65 nuclear translocation and pictures were taken at low and high (rectangle inserts) magnifications. The boundaries of corneal epithelium (epi) and stroma (str) were marked with dotted lines. The picture represents at least 3 slides of each mouse and 2 mice of each genotype used for the studies.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0016132-g002: IKKβ is dispensable for the maintenance of mouse corneal epithelium.The adult double (IkkβF/F) and triple (IkkβΔCE/ΔCE) transgenic mice were fed with Dox-chow for 30 days. (A) Genomic DNA was isolated from the corneal epithelial cells and subjected to PCR genotyping using primers specific for the IkkβΔ (deleted allele), IkkβF (floxed allele) and Gapdh. The triple transgenic mice lost IkkβF and acquired IkkβΔ alleles. (B) The eyes were photographed and (C) their tissue sections were subjected to histological analyses by H&E staining and molecular characterization by immunostaining (red) and nuclei were identified by DAPI staining (blue). The TUNEL assay was used to detect apoptosis, BrdU incorporation was used for proliferation, and the expression of KRT12 and KRT14 was used to evaluate corneal (KRT12) and basal (KRT14) epithelial differentiation. The activation of the IKK pathway was evaluated by p65 nuclear translocation and pictures were taken at low and high (rectangle inserts) magnifications. The boundaries of corneal epithelium (epi) and stroma (str) were marked with dotted lines. The picture represents at least 3 slides of each mouse and 2 mice of each genotype used for the studies.
Mentions: To evaluate the roles of IKKβ in maintenance of corneal homeostasis, we fed the adult double (Krt12rtTA/rtTA/IkkβF/F) and triple (Krt12rtTA/rtTA/tetO-Cre/IkkβF/F) transgenic mice Dox-chow for 30 days. To confirm the induction of Ikkβ gene deletion, we examined the genomic DNA isolated from corneal epithelial cells. By PCR, we detected only the IkkβF allele in cells isolated from double transgenic mice, whereas, we found only the IkkβΔ allele in cells isolated from triple transgenic mice (Fig. 2A). The triple transgenic mice with induced corneal epithelium-specific Ikkβ ablation are hereafter referred to as IkkβΔCE/ΔCE, whereas the control double transgenic mice are referred to as IkkβF/F.

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