Keratin 75 is a component of the LINC complex and has an essential role in mediating the SOX2 rapid healing response during wound repair

TO THE EDITOR

The wound healing process relies on the proper execution and regulation of signaling pathways and transcriptional regulators to properly coordinate the wound healing response (Eming et al., 2014). Although epithelial lining tissues have a remarkable ability for tissue repair, the efficiency of such repair varies depending on the tissue considered (Iglesias-Bartolome et al., 2018). For example, wounds of the oral mucosa heal more effectively than wounds of the skin (Iglesias-Bartolome et al., 2018). 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 (Iglesias-Bartolome et al., 2018). Reprogramming of cutaneous skin by SOX2 corresponded to increased expression of several wound-activated keratin genes including Krt6, Krt16 and Krt17 at baseline prior to wounding, further demonstrating priming of cutaneous skin for rapid wound healing (Iglesias-Bartolome et al., 2018, Uchiyama et al., 2019). In addition to the wound-activated keratin genes, we found SOX2 corresponded to increased expression of Krt75, 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 (Yang et al., 2019) and to be an essential component of the enamel organic matrix (Duverger et al., 2014; Chiba et al., 2019). Mutations in K75 had been previously shown to be directly correlated with defects in mouse hair shaft (Liu et al., 2016) and feather development in chicken (Ng et al., 2012). Therefore, the role of K75 extends beyond its structural support in hair, teeth and feather, and its role in interfollicular keratinocytes and wound healing is poorly understood.

To determine if SOX2 directly regulates Krt75 expression, we utilized our tamoxifen inducible K14creERTM/LSL-SOX2 mouse model (Iglesias-Bartolome et al., 2018, Uchiyama et al., 2019) and assessed K75 protein level by immunofluorescence staining. Induction of SOX2 by tamoxifen showed that K75 was highly expressed in the interfollicular epidermis compared to vehicle control skin, where it was primarily localized in hair follicles (Figure 1a). This was also confirmed by qPCR in which SOX2 significantly increased Krt75 expression (Figure 1b). Moreover, we identified three potential SOX2 binding sites (Sites 1, 2 and 3) in the Krt75 promoter, indicating direct transcriptional regulation of Krt75 by SOX2 (Figure 1c; Supplementary Table S1). We tested this by isolating primary mouse keratinocytes from our K14creERTM/LSL-SOX2 mouse model and induced SOX2 expression with tamoxifen treatment and performed ChIP-qPCR analysis. All three sites in the promoter of Krt75 were significantly enriched when pulled down using an anti-SOX2 antibody compared to IgG control (Figure 1d). These findings demonstrate Krt75 to be a direct target of SOX2.

Figure 1. Krt75 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). Scale bar=50μm. (b) qPCR of Krt75 from SOX2 transgenic mice treated with vehicle or tamoxifen. n=2. Data expressed as mean ± SD. *P<0.05, (c) Genome track of Krt75 gene demonstrating 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.

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 keratinocyte migration (Rotty and Coulombe, 2012, Wang et al., 2018). We transfected K75 construct in murine ameloblast-like cells (ALC), 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) (Ostlund et al., 2009). Keratins have been shown to interact with nuclear components and can be present in the nuclear interior (Hobbs et al., 2016). 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 (Carley et al., 2021, Ostlund et al., 2009, Stewart et al., 2015, Ueda et al., 2022), suggesting K75 interaction with LINC may regulate the wound healing response by regulating gene expression. To confirm mass spectrometry data, we performed proximity ligation assay (PLA) to confirm K75-SUN2 interaction in primary human keratinocytes overexpressing SOX2. Transduction efficiency of SOX2 was confirmed by immunofluorescence staining (Supplementary Figure S3). PLA 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 keratinocytes overexpressing SOX2 (Figure 2b and Supplementary Figure S4). Super-resolution imaging further demonstrated 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 keratinocytes (Supplementary Figure S4). Keratinocytes grown in culture are known to have a wound-activated phenotype that consists of induced expression of the wound-activated keratins Krt6, Krt16 and Krt17. Our data supports that cytoplasmic K75 interacts with SUN2 at the periphery of the nuclear envelope. These findings demonstrate K75 to be an additional marker of the wound-activated phenotype of keratinocytes and identify a previously unreported role for K75 as a component of the LINC complex.

Figure 2. K75 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). Scale bar= 5μm. Magnification of dashed box is shown on the bottom of the image. (b) Super resolution imaging of primary human keratinocytes overexpressing SOX2 to detect localization of K75 (green), SUN2 (red), and SOX2 (magenta). Magnification of dashed box is shown on the bottom of the image. (c) Western blot and quantification showing expression of K75, SUN2, 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. Rate of keratinocyte migration was quantified at 24 hours after scratch. n=3 per group. Data expressed as mean ± SEM. *P<0.05.

We next tested if the K75-SUN2 interaction is essential for the rapid wound healing effects mediated by SOX2. We utilized a keratinocyte scratch assay in which primary human keratinocytes were transduced with SOX2 and knockdown of K75 and SUN2 was performed with siRNA. 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 keratinocyte migration (Figure 2c and d). Knockdown of either K75 or SUN2 resulted in inhibition of keratinocyte migration and reversed SOX2 effects. Our findings describe K75 as a component of the LINC complex and has an essential role in mediating the SOX2 rapid healing response during wound healing.

Data availability statement

Datasets related to this article can be found at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE97615 and https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE118859, hosted at Gene Expression Omnibus GEO) (Iglesias-Bartolome et al., 2018, Uchiyama et al., 2019).