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Laboratory of Skin Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, USA
Laboratory of Skin Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, USA
Laboratory of Skin Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, USA
Laboratory of Skin Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, USA
Laboratory of Skin Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, USA
Laboratory of Skin Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, USA
Craniofacial Anomalies and Regeneration Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA
Laboratory of Skin Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, USA
The wound healing process relies on the proper execution and regulation of signaling pathways and transcriptional regulators to properly coordinate the wound healing response (
). Although epithelial lining tissues have a remarkable ability for tissue repair, the efficiency of such repair varies depending on the tissue considered (
). Recently, our laboratory performed a comparative analysis on human oral and cutaneous skin wounds and determined that a wound-activated transcriptional network regulated by the transcription factor SOX2 establishes a network that primes the oral epithelium for rapid wound repair (
). Reprogramming of cutaneous skin by SOX2 corresponded to increased expression of several wound-activated keratin genes, including keratin (K)6 gene K6, gene K16, and gene K17 at baseline before wounding, further showing priming of cutaneous skin for rapid wound healing (
). In addition to the wound-activated keratin genes, we found that SOX2 corresponded to increased expression of K75, a hair follicle type II keratin, in human oral mucosa and mouse models of rapid wound healing (Supplementary Figure S1), suggesting a role for this keratin in the SOX2-mediated rapid healing response. In addition to its role as a hair keratin gene, we identified K75 to be secreted by ameloblast cells to form dental enamel (
). Therefore, the role of K75 extends beyond its structural support in hair, teeth, and feather, and its role in interfollicular keratinocytes (KCs) and wound healing is poorly understood.
To determine whether SOX2 directly regulates K75 expression, we utilized our tamoxifen-inducible K14creERTM/LSL-SOX2 mouse model (
) and assessed K75 protein level by immunofluorescence staining. Induction of SOX2 by tamoxifen showed that K75 was highly expressed in the interfollicular epidermis compared with that in the vehicle control skin, where it was primarily localized in hair follicles (Figure 1a). This was also confirmed by qPCR in which SOX2 significantly increased K75 expression (Figure 1b). Moreover, we identified three potential SOX2-binding sites (sites 1, 2, and 3) in the K75 promoter, indicating direct transcriptional regulation of K75 by SOX2 (Figure 1c and Supplementary Table S1). We tested this by isolating primary mouse KCs from our K14creERTM/LSL-SOX2 mouse model and inducing SOX2 expression with tamoxifen treatment and performing chromatin immunoprecipitation qPCR analysis. All the three sites in the promoter of K75 were significantly enriched when pulled down using an anti-SOX2 antibody compared with those of IgG control (Figure 1d). These findings show K75 to be a direct target of SOX2.
Figure 1K75 is a direct transcriptional target of SOX2. (a) Immunofluorescence staining of unwounded skin tissue from SOX2-transgenic mice treated with vehicle or tamoxifen to detect K75 (red) and DAPI (blue). Bar = 50 μm. (b) qPCR of K75 from SOX2-transgenic mice treated with vehicle or tamoxifen. n = 2. Data expressed as mean ± SD. ∗P < 0.05, (c) Genome track of K75 gene showing three potential SOX2-binding sites (sites 1, 2, and 3). (d) qPCR analyses on chromatin samples from mouse primary keratinocytes isolated from SOX2-transgenic mice treated with tamoxifen or vehicle after immunoprecipitation with anti-SOX2 or IgG control antibodies. n = 2. Data expressed as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. chr, chromosome; K, keratin; Tam, tamoxifen; Veh, vehicle.
To further investigate the role of K75 in regulating the wound healing response, we identified potential protein binding partners by mass spectrometry. Protein interactions with keratins have been previously described and have shown to play an essential role in regulating KC migration (
). We transfected K75 construct in murine ameloblast-like cells, a type of oral epithelial cells, and found several binding partners for K75 (Supplementary Figure S2). Among them, we identified SUN2, an inner nuclear membrane protein and component of the linker of nucleoskeleton and cytoskeleton complex (LINC) (
). One of the main functions of LINC is to transduce mechanical signals from the plasma membrane to the nucleus where it can influence gene expression (
), suggesting that K75 interaction with LINC may regulate the wound healing response by regulating gene expression. To confirm mass spectrometry data, we performed a proximity ligation assay to confirm K75‒SUN2 interaction in primary human KCs overexpressing SOX2. Transduction efficiency of SOX2 was confirmed by immunofluorescence staining (Supplementary Figure S3). Proximity ligation assay confirmed K75‒SUN2 interaction, which was found to occur primarily at the cytoplasmic face of the nuclear periphery (Figure 2a). This was further confirmed by immunofluorescence staining in primary human KCs overexpressing SOX2 (Figure 2b and Supplementary Figure S4). Super-resolution imaging further showed cytoplasmic K75, and SUN2 detection was observed primarily at the nuclear periphery (Figure 2b and Supplementary Movie S1). Interestingly, we found K75 to be expressed in control KCs (Supplementary Figure S4). KCs grown in culture are known to have a wound-activated phenotype that consists of induced expression of the wound-activated keratins K6, K16, and K17. Our data support that cytoplasmic K75 interacts with SUN2 at the periphery of the nuclear envelope. These findings show K75 to be an additional marker of the wound-activated phenotype of KCs and identify a previously unreported role for K75 as a component of the LINC complex.
Figure 2K75 is a component of the LINC complex and is essential for promoting the SOX2-mediated rapid healing effects. (a) Proximity ligation assay of primary human keratinocytes overexpressing SOX2 to detect K75 and SUN2 (red) interaction and DAPI (blue). Bar = 5 μm. The magnification of the dashed box is shown at the bottom of the image. (b) Super-resolution imaging of primary human keratinocytes overexpressing SOX2 to detect the localization of K75 (green), SUN2 (red), and SOX2 (magenta). The magnification of the dashed box is shown at the bottom of the image. (c) Western blot and quantification showing the expression of K75, SUN2, and SOX2 in primary human keratinocytes after siRNA knockdown. RPS14 served as a loading control. (d) Scratch assay of primary human keratinocytes after siRNA of K75 and SUN2. The rate of keratinocyte migration was quantified 24 hours after the scratch. n = 3 per group. Data are expressed as mean ± SEM. ∗P < 0.05. K, keratin; LINC, linker of nucleoskeleton and cytoskeleton complex; siK75, small interfering RNA targeting K75; siNeg, small interfering RNA targeting negative control; siRNA, small interfering RNA; siSun2, small interfering RNA targeting SUN2.
We next tested whether the K75‒SUN2 interaction is essential for the rapid wound healing effects mediated by SOX2. We utilized a KC scratch assay in which primary human KCs were transduced with SOX2 and knockdown of K75 and SUN2 was performed with small interfering RNA. Knockdown efficiency of K75 and SUN2 was confirmed by western blot (Figure 2c). We show that overexpression of SOX2 resulted in increased K75 expression and KC migration (Figure 2c and d). Knockdown of either K75 or SUN2 resulted in inhibition of KC migration and reversed SOX2 effects. Our findings describe K75 as a component of the LINC complex and that it has an essential role in mediating the SOX2 rapid healing response during wound healing.
This work was supported by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health (ZIA-AR041124 to MIM). We thank the members of the National Institute of Arthritis and Musculoskeletal and Skin Diseases Light Imaging Core Facility.
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