Abstract
BackgroundKruppel-like factor KLF4 is a transcription factor critical for the establishment of the barrier function of the skin. Its function in stem cell biology has been recently recognized. Previous studies have revealed that hair follicle stem cells contribute to cutaneous wound healing. However, expression of KLF4 in hair follicle stem cells and the importance of such expression in cutaneous wound healing have not been investigated.Methodology/Principal FindingsQuantitative real time polymerase chain reaction (RT-PCR) analysis showed higher KLF4 expression in hair follicle stem cell-enriched mouse skin keratinocytes than that in control keratinocytes. We generated KLF4 promoter-driven enhanced green fluorescence protein (KLF4/EGFP) transgenic mice and tamoxifen-inducible KLF4 knockout mice by crossing KLF4 promoter-driven Cre recombinase fused with tamoxifen-inducible estrogen receptor (KLF4/CreER™) transgenic mice with KLF4(flox) mice. KLF4/EGFP cells purified from dorsal skin keratinocytes of KLF4/EGFP transgenic mice were co-localized with 5-bromo-2'-deoxyuridine (BrdU)-label retaining cells by flow cytometric analysis and immunohistochemistry. Lineage tracing was performed in the context of cutaneous wound healing, using KLF4/CreER™ and Rosa26RLacZ double transgenic mice, to examine the involvement of KLF4 in wound healing. We found that KLF4 expressing cells were likely derived from bulge stem cells. In addition, KLF4 expressing multipotent cells migrated to the wound and contributed to the wound healing. After knocking out KLF4 by tamoxifen induction of KLF4/CreER™ and KLF4(flox) double transgenic mice, we found that the population of bulge stem cell-enriched population was decreased, which was accompanied by significantly delayed cutaneous wound healing. Consistently, KLF4 knockdown by KLF4-specific small hairpin RNA in human A431 epidermoid carcinoma cells decreased the stem cell population and was accompanied by compromised cell migration.Conclusions/SignificanceKLF4 expression in mouse hair bulge stem cells plays an important role in cutaneous wound healing. These findings may enable future development of KLF4-based therapeutic strategies aimed at accelerating cutaneous wound closure.
Highlights
Skin is a continuously regenerating organ composed of a basal layer of proliferating cells and suprabasal layers of terminally differentiating cells that transit toward and are sloughed from skin surface [1]
Expression of Kruppel like factor 4 (KLF4) in mouse hair follicle stem cells To examine if KLF4 is enriched in mouse epidermal stem cells, we first purified these cells by fluorescent activated cell sorting from the skin of 6-week-old wild-type C57BL/6 mice and performed quantitative real time polymerase chain reaction (RT-PCR) analysis
CD34, G protein coupled receptor expressed in follicles (FEX) [39], gremlin, cysteine knot superfamily 1, BMP antagonist 1 (Cktsfb1), and dickkopf 3 (DDK3), highly expressed in CD34 enriched mouse keratinocyte stem cells, and KLF5 and bone morphogenesis protein 4 (Bmp4), highly expressed in differentiated cells [4], were used as controls
Summary
Skin is a continuously regenerating organ composed of a basal layer of proliferating cells and suprabasal layers of terminally differentiating cells that transit toward and are sloughed from skin surface [1]. Epidermal renewal is thought to be controlled by stem cells located either in the basal layer of the interfollicular epidermis (IFE) or in the deepest portion of permanent hair follicle called bulge [2]. Previous work has reported that bulge stem cells rapidly respond to wounding and migrate towards the IFE to help with the rapid hair-follicle regeneration and that bulge-derived cells are transient amplifying cells committed to differentiation [9,12,14]. The role and contribution of keratinocytes derived from hair follicle bulge stem cells to cutaneous wound healing need further elucidation. Previous studies have revealed that hair follicle stem cells contribute to cutaneous wound healing. Expression of KLF4 in hair follicle stem cells and the importance of such expression in cutaneous wound healing have not been investigated
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