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I?B kinase ? regulates epithelium migration during corneal wound healing.

ABSTRACT: The IKK? is known to regulate transcription factor NF-?B activation leading to inflammatory responses. Recent gene knockout studies have shown that IKK? can orchestrate local inflammatory responses and regulate homeostasis of epithelial tissues. To investigate whether IKK? 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/Ikk?(F/F) (Ikk?(?CE/?CE)) 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 Ikk?(F/F) mice. Doxycycline-induced IKK? ablation occurred in corneal epithelial cells of triple transgenic Ikk?(?CE/?CE) mice, but loss of IKK? did not cause ocular abnormalities in fetal development and postnatal maintenance. Instead, loss of IKK? 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 IKK? was required for cytokine-induced cell migration and wound closure but was dispensable for cell proliferation. In both in vivo and in vitro settings, IKK? was required for optimal activation of NF-?B and p38 signaling in corneal epithelial cells, and p38 activation is likely mediated through formation of an IKK?-p38 protein complex. Thus, our studies in corneal epithelium reveal a previously un-recognized role for IKK? in the control of epithelial cell motility and wound healing.

SUBMITTER: Chen L

PROVIDER: S-EPMC3022035 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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IκB kinase β regulates epithelium migration during corneal wound healing.

Chen Liang L Meng Qinghang Q Kao Winston W Xia Ying Y

PloS one 20110117 1

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 ...[more]

PMID: 21264230

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Project description:The aim of this study was to determine whether bone marrow-derived fibrocytes migrate into the cornea after stromal scar-producing injury and differentiate into alpha-smooth muscle actin (?SMA) + myofibroblasts. Chimeric mice expressing green fluorescent protein (GFP) bone marrow cells had fibrosis (haze)-generating irregular phototherapeutic keratectomy (PTK). Multiplex immunohistochemistry (IHC) for GFP and fibrocyte markers (CD34, CD45, and vimentin) was used to detect fibrocyte infiltration into the corneal stroma and the development of GFP+ ?SMA+ myofibroblasts. IHC for activated caspase-3, GFP and CD45 was used to detect fibrocyte and other hematopoietic cells undergoing apoptosis. Moderate haze developed in PTK-treated mouse corneas at 14 days after surgery and worsened, and persisted, at 21 days after surgery. GFP+ CD34+ CD45+ fibrocytes, likely in addition to other CD34+ and/or CD45+ hematopoietic and stem/progenitor cells, infiltrated the cornea and were present in the stroma in high numbers by one day after PTK. The fibrocytes and other bone marrow-derived cells progressively decreased at four days and seven days after surgery. At four days after PTK, 5% of the GFP+ cells expressed activated caspase-3. At 14 days after PTK, more than 50% of GFP+ CD45+ cells were also ?SMA+ myofibroblasts. At 21 days after PTK, few GFP+ ?SMA+ cells persisted in the stroma and more than 95% of those remaining expressed activated caspase-3, indicating they were undergoing apoptosis. GFP+ CD45+ SMA+ cells that developed from 4 to 21 days after irregular PTK were likely developed from fibrocytes. After irregular PTK in the strain of C57BL/6-C57/BL/6-Tg(UBC-GFP)30Scha/J chimeric mice, however, more than 95% of fibrocytes and other hematopoietic cells underwent apoptosis prior to the development of mature ?SMA+ myofibroblasts. Most GFP+ CD45+ ?SMA+ myofibroblasts that did develop subsequently underwent apoptosis-likely due to epithelial basement membrane regeneration and deprivation of epithelium-derived TGF? requisite for myofibroblast survival.

Project description:BackgroundThe mouse corneal epithelium is a continuously renewing 5-6 cell thick protective layer covering the corneal surface, which regenerates rapidly when injured. It is maintained by peripherally located limbal stem cells (LSCs) that produce transient amplifying cells (TACs) which proliferate, migrate centripetally, differentiate and are eventually shed from the epithelial surface. LSC activity is required both for normal tissue maintenance and wound healing. Mosaic analysis can provide insights into LSC function, cell movement and cell mixing during tissue maintenance and repair. The present study investigates cell streaming during corneal maintenance and repair and changes in LSC function with age.ResultsThe initial pattern of corneal epithelial patches in XLacZ+/- X-inactivation mosaics was replaced after birth by radial stripes, indicating activation of LSCs. Stripe patterns (clockwise, anticlockwise or midline) were independent between paired eyes. Wound healing in organ culture was analysed by mosaic analysis of XLacZ+/- eyes or time-lapse imaging of GFP mosaics. Both central and peripheral wounds healed clonally, with cells moving in from all around the wound circumference without significant cell mixing, to reconstitute striping patterns. Mosaic analysis revealed that wounds can heal asymmetrically. Healing of peripheral wounds produced stripe patterns that mimicked some aberrant striping patterns observed in unwounded corneas. Quantitative analysis provided no evidence for an uneven distribution of LSC clones but showed that corrected corneal epithelial stripe numbers declined with age (implying declining LSC function) but stabilised after 39 weeks.ConclusionStriping patterns, produced by centripetal movement, are defined independently and stochastically in individual eyes. Little cell mixing occurs during the initial phase of wound healing and the direction of cell movement is determined by the position of the wound and not by population pressure from the limbus. LSC function declines with age and this may reflect reduced LSCs numbers, more quiescent LSCs or a reduced ability of older stem cells to maintain tissue homeostasis. The later plateau of LSC function might indicate the minimum LSC function that is sufficient for corneal epithelial maintenance. Quantitative and temporal mosaic analyses provide new possibilities for studying stem cell function, tissue maintenance and repair.

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I?B kinase ? regulates epithelium migration during corneal wound healing.

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IκB kinase β regulates epithelium migration during corneal wound healing.

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