Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, Columbia, South Carolina, United States of America.
PLoS One. 2012;7(6):e39663. doi: 10.1371/journal.pone.0039663. Epub 2012 Jun 20.
Kruppel-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 FINDINGS: Quantitative 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/SIGNIFICANCE: KLF4 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.
Kruppel 样因子 KLF4 是一个对皮肤屏障功能的建立至关重要的转录因子。其在干细胞生物学中的功能最近才被认识到。先前的研究表明，毛囊干细胞有助于皮肤伤口愈合。然而，毛囊干细胞中 KLF4 的表达及其在皮肤伤口愈合中的重要性尚未得到研究。
方法/主要发现：定量实时聚合酶链反应 (RT-PCR) 分析显示，富含毛囊干细胞的小鼠皮肤角质细胞中的 KLF4 表达高于对照角质细胞。我们通过将 KLF4 启动子驱动的 Cre 重组酶与他莫昔芬诱导型雌激素受体 (KLF4/CreER™) 转基因小鼠与 KLF4(flox) 小鼠杂交，生成 KLF4 启动子驱动的增强型绿色荧光蛋白 (KLF4/EGFP) 转基因小鼠和他莫昔芬诱导型 KLF4 敲除小鼠。通过流式细胞术分析和免疫组织化学，从 KLF4/EGFP 转基因小鼠的背部皮肤角质细胞中纯化 KLF4/EGFP 细胞，与 BrdU-标记保留细胞共定位。在皮肤伤口愈合的背景下进行谱系追踪，使用 KLF4/CreER™ 和 Rosa26RLacZ 双转基因小鼠，以检查 KLF4 在伤口愈合中的参与情况。我们发现，表达 KLF4 的细胞可能来自隆起干细胞。此外，表达 KLF4 的多能细胞迁移到伤口处，并有助于伤口愈合。通过他莫昔芬诱导 KLF4/CreER™ 和 KLF4(flox) 双转基因小鼠敲除 KLF4 后，我们发现隆起干细胞丰富的群体减少，这伴随着皮肤伤口愈合明显延迟。一致地，在人 A431 表皮癌细胞中用 KLF4 特异性短发夹 RNA 敲低 KLF4 降低了干细胞群体，并伴随着细胞迁移受损。
结论/意义：KLF4 在小鼠毛囊隆起干细胞中的表达在皮肤伤口愈合中起着重要作用。这些发现可能为未来基于 KLF4 的治疗策略的发展提供了依据，旨在加速皮肤伤口闭合。
