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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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The IKKβ and p38 form complexes in vitro and in vivo.The hTCEpi cells were either untreated or treated with TNF-α (10 ng/ml) for 0.5 hour or as indicated. In some experiments, chemical inhibitors for p38, SB202190 (1 µM), JNK, SP600125 (1 µM), ERK, PD98059 (5 µM), and IKK, JSH23 (1 µM), were used 0.5 hour prior to TNF-α. Cell lysates were subjected to (A) pull-down assays using GST-p38 and glutathione-agarose beads, and (B) immunoprecipitation using anti-p38, anti-IKKβ and anti-MKK6 antibodies. The pull-down/precipitated proteins and total cell lysates were analyzed by Western blotting using antibodies as indicated. (C) The cell lysates were analyzed by Western blotting for phospho-ATF2 and -p65, and total IκBα and β-Actin. The relative fold induction was calculated based on the intensity of the bands in control (set as 1) and TNF-α treated samples. (D) The hTCEpi cells were either untreated or pre-treated with various chemical inhibitors as indicated for 0.5 hour. The cells were subjected to in vitro scratch wound healing assay in the presence or absence of TNF-α (10 ng/ml) for 48 hours. The wound closure rates were calculated based on the mean ± SD of 4 repeats and statistical analyses were done by comparing to the rates in control cells. *: p<0.05; **: p<0.01; ***: p<0.001.
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pone-0016132-g006: The IKKβ and p38 form complexes in vitro and in vivo.The hTCEpi cells were either untreated or treated with TNF-α (10 ng/ml) for 0.5 hour or as indicated. In some experiments, chemical inhibitors for p38, SB202190 (1 µM), JNK, SP600125 (1 µM), ERK, PD98059 (5 µM), and IKK, JSH23 (1 µM), were used 0.5 hour prior to TNF-α. Cell lysates were subjected to (A) pull-down assays using GST-p38 and glutathione-agarose beads, and (B) immunoprecipitation using anti-p38, anti-IKKβ and anti-MKK6 antibodies. The pull-down/precipitated proteins and total cell lysates were analyzed by Western blotting using antibodies as indicated. (C) The cell lysates were analyzed by Western blotting for phospho-ATF2 and -p65, and total IκBα and β-Actin. The relative fold induction was calculated based on the intensity of the bands in control (set as 1) and TNF-α treated samples. (D) The hTCEpi cells were either untreated or pre-treated with various chemical inhibitors as indicated for 0.5 hour. The cells were subjected to in vitro scratch wound healing assay in the presence or absence of TNF-α (10 ng/ml) for 48 hours. The wound closure rates were calculated based on the mean ± SD of 4 repeats and statistical analyses were done by comparing to the rates in control cells. *: p<0.05; **: p<0.01; ***: p<0.001.

Mentions: The molecular connection of IKKβ to NF-κB, based on direct interaction and phosphorylation of IκBα, is well established, but the link to p38 remains obscure. To look into the molecular basis of the latter, we examined the physical interactions between IKKβ and p38. The hTCEpi cells were either un-treated or treated with TNF-α for 20 min to induce an apparent IκBα degradation and p65 phosphorylation, indicative of the NF-κB pathway activation (Figs. 6A and 6B). From both un-treated and TNF-α-treated hTCEpi cells, the GST-p38 and anti-p38 antibodies were able to pull down IKKβ (Figs. 6A and 6B). The p38 is a mitogen-activated protein kinase, known to interact with and be phosphorylated by its upstream kinases, MKK3, MKK4 and MKK6, in response to mitogenic and stress stimuli [38]. Antibodies to MKK6, however, were unable to co-precipitate IKKβ, indicating that the IKKβ-p38 complexes were independent of MKK6. We suggest that the IKKβ-p38 complexes are distinct from the MKK6-p38 and are used primarily for effective and specific transduction of cytokine signals in hTCEpi cells (Fig. 5).


IκB kinase β regulates epithelium migration during corneal wound healing.

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

The IKKβ and p38 form complexes in vitro and in vivo.The hTCEpi cells were either untreated or treated with TNF-α (10 ng/ml) for 0.5 hour or as indicated. In some experiments, chemical inhibitors for p38, SB202190 (1 µM), JNK, SP600125 (1 µM), ERK, PD98059 (5 µM), and IKK, JSH23 (1 µM), were used 0.5 hour prior to TNF-α. Cell lysates were subjected to (A) pull-down assays using GST-p38 and glutathione-agarose beads, and (B) immunoprecipitation using anti-p38, anti-IKKβ and anti-MKK6 antibodies. The pull-down/precipitated proteins and total cell lysates were analyzed by Western blotting using antibodies as indicated. (C) The cell lysates were analyzed by Western blotting for phospho-ATF2 and -p65, and total IκBα and β-Actin. The relative fold induction was calculated based on the intensity of the bands in control (set as 1) and TNF-α treated samples. (D) The hTCEpi cells were either untreated or pre-treated with various chemical inhibitors as indicated for 0.5 hour. The cells were subjected to in vitro scratch wound healing assay in the presence or absence of TNF-α (10 ng/ml) for 48 hours. The wound closure rates were calculated based on the mean ± SD of 4 repeats and statistical analyses were done by comparing to the rates in control cells. *: p<0.05; **: p<0.01; ***: p<0.001.
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pone-0016132-g006: The IKKβ and p38 form complexes in vitro and in vivo.The hTCEpi cells were either untreated or treated with TNF-α (10 ng/ml) for 0.5 hour or as indicated. In some experiments, chemical inhibitors for p38, SB202190 (1 µM), JNK, SP600125 (1 µM), ERK, PD98059 (5 µM), and IKK, JSH23 (1 µM), were used 0.5 hour prior to TNF-α. Cell lysates were subjected to (A) pull-down assays using GST-p38 and glutathione-agarose beads, and (B) immunoprecipitation using anti-p38, anti-IKKβ and anti-MKK6 antibodies. The pull-down/precipitated proteins and total cell lysates were analyzed by Western blotting using antibodies as indicated. (C) The cell lysates were analyzed by Western blotting for phospho-ATF2 and -p65, and total IκBα and β-Actin. The relative fold induction was calculated based on the intensity of the bands in control (set as 1) and TNF-α treated samples. (D) The hTCEpi cells were either untreated or pre-treated with various chemical inhibitors as indicated for 0.5 hour. The cells were subjected to in vitro scratch wound healing assay in the presence or absence of TNF-α (10 ng/ml) for 48 hours. The wound closure rates were calculated based on the mean ± SD of 4 repeats and statistical analyses were done by comparing to the rates in control cells. *: p<0.05; **: p<0.01; ***: p<0.001.
Mentions: The molecular connection of IKKβ to NF-κB, based on direct interaction and phosphorylation of IκBα, is well established, but the link to p38 remains obscure. To look into the molecular basis of the latter, we examined the physical interactions between IKKβ and p38. The hTCEpi cells were either un-treated or treated with TNF-α for 20 min to induce an apparent IκBα degradation and p65 phosphorylation, indicative of the NF-κB pathway activation (Figs. 6A and 6B). From both un-treated and TNF-α-treated hTCEpi cells, the GST-p38 and anti-p38 antibodies were able to pull down IKKβ (Figs. 6A and 6B). The p38 is a mitogen-activated protein kinase, known to interact with and be phosphorylated by its upstream kinases, MKK3, MKK4 and MKK6, in response to mitogenic and stress stimuli [38]. Antibodies to MKK6, however, were unable to co-precipitate IKKβ, indicating that the IKKβ-p38 complexes were independent of MKK6. We suggest that the IKKβ-p38 complexes are distinct from the MKK6-p38 and are used primarily for effective and specific transduction of cytokine signals in hTCEpi cells (Fig. 5).

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