Real-time monitoring of keratin 5 expression during burn re-epithelialization.
ABSTRACT Keratin is a major protein produced during epithelialization following burn injury and is a useful marker for assessing wound healing. Transgenic mice expressing enhanced green fluorescent protein (EGFP) driven by the keratin 5 (K5) promoter (K5GFP mice) were used to monitor keratin expression, and thus, re-epithelialization of burn wounds.
K5GFP transgenic mice were created using conventional techniques, with PCR and Southern blot confirmation of transgene incorporation, followed by selection of the line with the most intense and consistent basal epithelial EGFP expression. Epi-fluorescent microscopy of 24 K5GFP mouse flanks and 10 negative littermate controls was used to characterize EGFP intensity, before wounding and serially for 30 days after administration of a standardized burn wound and excision. Biopsy sections of K5GFP and negative control mice were stained with K5 antibody and imaged with confocal microscopy to characterize the distribution of EGFP and K5 at baseline and after injury and to examine the correlation between K5 expression and EGFP expression during healing.
Green fluorescence intensity increased at the advancing wound margin of burned K5GFP mice, reaching a maximum between days 12 and 15 post-burn and then decreasing as healing completed. K5 and EGFP expression increased in parallel in burned K5GFP mice as demonstrated by confocal microscopy.
EGFP expression correlated with K5 expression during wound healing and therefore serves as a good marker of re-epithelialization. This transgenic model allows noninvasive, real-time assessment of in vivo K5 expression and will be useful in the study of wound healing.
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ABSTRACT: Significant damage to tissue surrounding burn injuries occurs after the removal of the thermal source. This damage is caused by a combination of both necrotic and apoptotic cell death in the zone of stasis. Preserving the zone of stasis can reduce the wound size and thereby improve wound healing. We tested whether a peptide previously identified to inhibit necrotic and apoptotic cell death in neurons through c-Jun inhibition could enhance wound healing. We first tested the effects of this peptide on a keratinocyte and fibroblast cell line in culture. The peptide promoted proliferation of keratinocytes but had no effect on fibroblast proliferation, while the peptide also inhibited ultraviolet-induced apoptosis of keratinocytes. We finally tested the peptide in vivo, using a mouse model of burn injury. Wounds that were treated with the peptide reepithelialized faster than controls, while cell death surrounding the wound site was markedly reduced 24 hours postinjury, suggesting that the prevention of apoptosis as well as the proliferative effects of this peptide contribute to the wound healing process. Our data implicate c-Jun in multiple processes during wound repair and demonstrate that treatment of burn injuries using inhibitors of c-Jun dimerization at the time of injury can promote wound healing.Wound Repair and Regeneration 16(1):58-64. · 2.91 Impact Factor
Article: Exploiting the keratin 17 gene promoter to visualize live cells in epithelial appendages of mice.[show abstract] [hide abstract]
ABSTRACT: Keratin genes afford, given their large number (>50) and differential regulation, a unique opportunity to study the mechanisms underlying specification and differentiation in epithelia of higher metazoans. Moreover, the small size and regulation in cis of many keratin genes enable the use of their regulatory sequence to achieve targeted gene expression in mice. Here we show that 2 kilobases of 5' upstream region from the mouse keratin 17 gene (mK17) confers expression of green fluorescent protein (GFP) in major epithelial appendages of transgenic mice. Like that of mK17, onset of [mK17 5']-GFP reporter expression coincides with the appearance of ectoderm-derived epithelial appendages during embryonic development. In adult mice, [mK17 5']-GFP is appropriately regulated within hair, nail, glands, and oral papilla. Tracking of GFP fluorescence allows for the visualization of growth cycle-related changes in hair follicles, and the defects engendered by the hairless mutation, in live skin tissue. Deletion of an internal 48-bp interval, which encompasses a Gli-responsive element, from this promoter results in loss of GFP fluorescence in most appendages in vivo, suggesting that sonic hedgehog participates in K17 regulation. The compact mK17 gene promoter provides a novel tool for appendage-preferred gene expression and manipulation in transgenic mice.Molecular and Cellular Biology 09/2005; 25(16):7249-59. · 5.53 Impact Factor