Keratin 75 Is a Component of the LINC Complex and Has an Essential Role in Mediating the SOX2 Rapid Healing Response during Wound Repair (2022)

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., 2014Eming S.A. Martin P. Tomic-Canic M. Wound repair and regeneration: mechanisms, signaling, and translation.Sci Transl Med. 2014; 6: 265sr6Crossref PubMed Scopus (1428) Google Scholar). 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., 2018Iglesias-Bartolome R. Uchiyama A. Molinolo A.A. Abusleme L. Brooks S.R. Callejas-Valera J.L. et al.Transcriptional signature primes human oral mucosa for rapid wound healing.Sci Transl Med. 2018; 10eaap8798Crossref PubMed Scopus (83) Google Scholar). For example, wounds of the oral mucosa heal more effectively than wounds of the skin (Iglesias-Bartolome et al., 2018Iglesias-Bartolome R. Uchiyama A. Molinolo A.A. Abusleme L. Brooks S.R. Callejas-Valera J.L. et al.Transcriptional signature primes human oral mucosa for rapid wound healing.Sci Transl Med. 2018; 10eaap8798Crossref PubMed Scopus (83) Google Scholar). 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., 2018Iglesias-Bartolome R. Uchiyama A. Molinolo A.A. Abusleme L. Brooks S.R. Callejas-Valera J.L. et al.Transcriptional signature primes human oral mucosa for rapid wound healing.Sci Transl Med. 2018; 10eaap8798Crossref PubMed Scopus (83) Google Scholar). 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 (Iglesias-Bartolome et al., 2018Iglesias-Bartolome R. Uchiyama A. Molinolo A.A. Abusleme L. Brooks S.R. Callejas-Valera J.L. et al.Transcriptional signature primes human oral mucosa for rapid wound healing.Sci Transl Med. 2018; 10eaap8798Crossref PubMed Scopus (83) Google Scholar; Uchiyama et al., 2019Uchiyama A. Nayak S. Graf R. Cross M. Hasneen K. Gutkind J.S. et al.SOX2 epidermal overexpression promotes cutaneous wound healing via activation of EGFR/MEK/ERK signaling mediated by EGFR ligands.J Invest Dermatol. 2019; 139: 1809-1820.e8Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). 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 (Yang et al., 2019Yang X. Yamazaki H. Yamakoshi Y. Duverger O. Morasso M.I. Beniash E. Trafficking and secretion of keratin 75 by ameloblasts in vivo.J Biol Chem. 2019; 294: 18475-18487Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar) and to be an essential component of the enamel organic matrix (Chiba et al., 2019Chiba R. Okubo M. Yamamoto R. Saito M.M. Kobayashi S. Beniash E. et al.Porcine keratin 75 in developing enamel.J Oral Biosci. 2019; 61: 163-172Crossref PubMed Scopus (1) Google Scholar; Duverger et al., 2014Duverger O. Ohara T. Shaffer J.R. Donahue D. Zerfas P. Dullnig A. et al.Hair keratin mutations in tooth enamel increase dental decay risk.J Clin Invest. 2014; 124: 5219-5224Crossref PubMed Scopus (34) Google Scholar). Sequence variations in K75 had been previously shown to be directly correlated with defects in mouse hair shafts (Liu et al., 2016Liu Y. Snedecor E.R. Zhang X. Xu Y. Huang L. Jones E.C. et al.Correction of hair shaft defects through allele-specific silencing of mutant Krt75.J Invest Dermatol. 2016; 136: 45-51Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar) and feather development in chicken (Ng et al., 2012Ng C.S. Wu P. Foley J. McDonald M.L. Juan W.T. Huang C.J. et al.The chicken frizzle feather is due to an α-keratin (KRT75) mutation that causes a defective rachis.PLoS Genet. 2012; 8e1002748Crossref Scopus (71) Google Scholar). 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 (Iglesias-Bartolome et al., 2018Iglesias-Bartolome R. Uchiyama A. Molinolo A.A. Abusleme L. Brooks S.R. Callejas-Valera J.L. et al.Transcriptional signature primes human oral mucosa for rapid wound healing.Sci Transl Med. 2018; 10eaap8798Crossref PubMed Scopus (83) Google Scholar; Uchiyama et al., 2019Uchiyama A. Nayak S. Graf R. Cross M. Hasneen K. Gutkind J.S. et al.SOX2 epidermal overexpression promotes cutaneous wound healing via activation of EGFR/MEK/ERK signaling mediated by EGFR ligands.J Invest Dermatol. 2019; 139: 1809-1820.e8Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) 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.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 (Rotty and Coulombe, 2012Rotty J.D. Coulombe P.A. A wound-induced keratin inhibits Src activity during keratinocyte migration and tissue repair.J Cell Biol. 2012; 197: 381-389Crossref PubMed Scopus (70) Google Scholar; Wang et al., 2018Wang F. Chen S. Liu H.B. Parent C.A. Coulombe P.A. Keratin 6 regulates collective keratinocyte migration by altering cell-cell and cell-matrix adhesion.J Cell Biol. 2018; 217: 4314-4330Crossref PubMed Scopus (38) Google Scholar). 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) (Ostlund et al., 2009Ostlund C. Folker E.S. Choi J.C. Gomes E.R. Gundersen G.G. Worman H.J. Dynamics and molecular interactions of linker of nucleoskeleton and cytoskeleton (LINC) complex proteins.J Cell Sci. 2009; 122: 4099-4108Crossref PubMed Scopus (124) Google Scholar). Keratins have been shown to interact with nuclear components and can be present in the nuclear interior (Hobbs et al., 2016Hobbs R.P. Jacob J.T. Coulombe P.A. Keratins are going nuclear.Dev Cell. 2016; 38: 227-233Abstract Full Text Full Text PDF PubMed Google Scholar). 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., 2021Carley E. Stewart R.M. Zieman A. Jalilian I. King D.E. Zubek A. et al.The LINC complex transmits integrin-dependent tension to the nuclear lamina and represses epidermal differentiation.ELife. 2021; 10Crossref PubMed Scopus (12) Google Scholar; Ostlund et al., 2009Ostlund C. Folker E.S. Choi J.C. Gomes E.R. Gundersen G.G. Worman H.J. Dynamics and molecular interactions of linker of nucleoskeleton and cytoskeleton (LINC) complex proteins.J Cell Sci. 2009; 122: 4099-4108Crossref PubMed Scopus (124) Google Scholar; Stewart et al., 2015Stewart R.M. Zubek A.E. Rosowski K.A. Schreiner S.M. Horsley V. King M.C. Nuclear-cytoskeletal linkages facilitate cross talk between the nucleus and intercellular adhesions.J Cell Biol. 2015; 209: 403-418Crossref PubMed Scopus (44) Google Scholar; Ueda et al., 2022Ueda N. Maekawa M. Matsui T.S. Deguchi S. Takata T. Katahira J. et al.Inner nuclear membrane protein, sun1, is required for cytoskeletal force generation and focal adhesion maturation.Front Cell Dev Biol. 2022; 10885859Crossref PubMed Scopus (1) Google Scholar), 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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.Data availability statementDatasets 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 (Iglesias-Bartolome et al., 2018Iglesias-Bartolome R. Uchiyama A. Molinolo A.A. Abusleme L. Brooks S.R. Callejas-Valera J.L. et al.Transcriptional signature primes human oral mucosa for rapid wound healing.Sci Transl Med. 2018; 10eaap8798Crossref PubMed Scopus (83) Google Scholar; Uchiyama et al., 2019Uchiyama A. Nayak S. Graf R. Cross M. Hasneen K. Gutkind J.S. et al.SOX2 epidermal overexpression promotes cutaneous wound healing via activation of EGFR/MEK/ERK signaling mediated by EGFR ligands.J Invest Dermatol. 2019; 139: 1809-1820.e8Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar).Ethics statementAll procedures were approved by the National Institute of Arthritis and Musculoskeletal and Skin Diseases Animal Care and Use Committee.ORCIDsAndrew P. Sawaya: http://orcid.org/0000-0002-4675-3919Akihiko Uchiyama: http://orcid.org/0000-0003-0357-2808Emma Hope: http://orcid.org/0000-0002-9114-424XDeepti Bajpai: http://orcid.org/0000-0001-5701-8218Stephen Worrell: http://orcid.org/0000-0002-3605-7135Michael Cross: http://orcid.org/0000-0002-2446-9167Elia Beniash: http://orcid.org/0000-0001-9019-5160Lisa Jenkins: http://orcid.org/0000-0003-1245-1338Olivier Duverger: http://orcid.org/0000-0003-3200-1019Maria I. Morasso: http://orcid.org/0000-0002-9632-9593Conflict of InterestThe authors state no conflict of interest. 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., 2014Eming S.A. Martin P. Tomic-Canic M. Wound repair and regeneration: mechanisms, signaling, and translation.Sci Transl Med. 2014; 6: 265sr6Crossref PubMed Scopus (1428) Google Scholar). 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., 2018Iglesias-Bartolome R. Uchiyama A. Molinolo A.A. Abusleme L. Brooks S.R. Callejas-Valera J.L. et al.Transcriptional signature primes human oral mucosa for rapid wound healing.Sci Transl Med. 2018; 10eaap8798Crossref PubMed Scopus (83) Google Scholar). For example, wounds of the oral mucosa heal more effectively than wounds of the skin (Iglesias-Bartolome et al., 2018Iglesias-Bartolome R. Uchiyama A. Molinolo A.A. Abusleme L. Brooks S.R. Callejas-Valera J.L. et al.Transcriptional signature primes human oral mucosa for rapid wound healing.Sci Transl Med. 2018; 10eaap8798Crossref PubMed Scopus (83) Google Scholar). 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., 2018Iglesias-Bartolome R. Uchiyama A. Molinolo A.A. Abusleme L. Brooks S.R. Callejas-Valera J.L. et al.Transcriptional signature primes human oral mucosa for rapid wound healing.Sci Transl Med. 2018; 10eaap8798Crossref PubMed Scopus (83) Google Scholar). 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 (Iglesias-Bartolome et al., 2018Iglesias-Bartolome R. Uchiyama A. Molinolo A.A. Abusleme L. Brooks S.R. Callejas-Valera J.L. et al.Transcriptional signature primes human oral mucosa for rapid wound healing.Sci Transl Med. 2018; 10eaap8798Crossref PubMed Scopus (83) Google Scholar; Uchiyama et al., 2019Uchiyama A. Nayak S. Graf R. Cross M. Hasneen K. Gutkind J.S. et al.SOX2 epidermal overexpression promotes cutaneous wound healing via activation of EGFR/MEK/ERK signaling mediated by EGFR ligands.J Invest Dermatol. 2019; 139: 1809-1820.e8Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). 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 (Yang et al., 2019Yang X. Yamazaki H. Yamakoshi Y. Duverger O. Morasso M.I. Beniash E. Trafficking and secretion of keratin 75 by ameloblasts in vivo.J Biol Chem. 2019; 294: 18475-18487Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar) and to be an essential component of the enamel organic matrix (Chiba et al., 2019Chiba R. Okubo M. Yamamoto R. Saito M.M. Kobayashi S. Beniash E. et al.Porcine keratin 75 in developing enamel.J Oral Biosci. 2019; 61: 163-172Crossref PubMed Scopus (1) Google Scholar; Duverger et al., 2014Duverger O. Ohara T. Shaffer J.R. Donahue D. Zerfas P. Dullnig A. et al.Hair keratin mutations in tooth enamel increase dental decay risk.J Clin Invest. 2014; 124: 5219-5224Crossref PubMed Scopus (34) Google Scholar). Sequence variations in K75 had been previously shown to be directly correlated with defects in mouse hair shafts (Liu et al., 2016Liu Y. Snedecor E.R. Zhang X. Xu Y. Huang L. Jones E.C. et al.Correction of hair shaft defects through allele-specific silencing of mutant Krt75.J Invest Dermatol. 2016; 136: 45-51Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar) and feather development in chicken (Ng et al., 2012Ng C.S. Wu P. Foley J. McDonald M.L. Juan W.T. Huang C.J. et al.The chicken frizzle feather is due to an α-keratin (KRT75) mutation that causes a defective rachis.PLoS Genet. 2012; 8e1002748Crossref Scopus (71) Google Scholar). 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 (Iglesias-Bartolome et al., 2018Iglesias-Bartolome R. Uchiyama A. Molinolo A.A. Abusleme L. Brooks S.R. Callejas-Valera J.L. et al.Transcriptional signature primes human oral mucosa for rapid wound healing.Sci Transl Med. 2018; 10eaap8798Crossref PubMed Scopus (83) Google Scholar; Uchiyama et al., 2019Uchiyama A. Nayak S. Graf R. Cross M. Hasneen K. Gutkind J.S. et al.SOX2 epidermal overexpression promotes cutaneous wound healing via activation of EGFR/MEK/ERK signaling mediated by EGFR ligands.J Invest Dermatol. 2019; 139: 1809-1820.e8Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) 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. 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 (Rotty and Coulombe, 2012Rotty J.D. Coulombe P.A. A wound-induced keratin inhibits Src activity during keratinocyte migration and tissue repair.J Cell Biol. 2012; 197: 381-389Crossref PubMed Scopus (70) Google Scholar; Wang et al., 2018Wang F. Chen S. Liu H.B. Parent C.A. Coulombe P.A. Keratin 6 regulates collective keratinocyte migration by altering cell-cell and cell-matrix adhesion.J Cell Biol. 2018; 217: 4314-4330Crossref PubMed Scopus (38) Google Scholar). 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) (Ostlund et al., 2009Ostlund C. Folker E.S. Choi J.C. Gomes E.R. Gundersen G.G. Worman H.J. Dynamics and molecular interactions of linker of nucleoskeleton and cytoskeleton (LINC) complex proteins.J Cell Sci. 2009; 122: 4099-4108Crossref PubMed Scopus (124) Google Scholar). Keratins have been shown to interact with nuclear components and can be present in the nuclear interior (Hobbs et al., 2016Hobbs R.P. Jacob J.T. Coulombe P.A. Keratins are going nuclear.Dev Cell. 2016; 38: 227-233Abstract Full Text Full Text PDF PubMed Google Scholar). 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., 2021Carley E. Stewart R.M. Zieman A. Jalilian I. King D.E. Zubek A. et al.The LINC complex transmits integrin-dependent tension to the nuclear lamina and represses epidermal differentiation.ELife. 2021; 10Crossref PubMed Scopus (12) Google Scholar; Ostlund et al., 2009Ostlund C. Folker E.S. Choi J.C. Gomes E.R. Gundersen G.G. Worman H.J. Dynamics and molecular interactions of linker of nucleoskeleton and cytoskeleton (LINC) complex proteins.J Cell Sci. 2009; 122: 4099-4108Crossref PubMed Scopus (124) Google Scholar; Stewart et al., 2015Stewart R.M. Zubek A.E. Rosowski K.A. Schreiner S.M. Horsley V. King M.C. Nuclear-cytoskeletal linkages facilitate cross talk between the nucleus and intercellular adhesions.J Cell Biol. 2015; 209: 403-418Crossref PubMed Scopus (44) Google Scholar; Ueda et al., 2022Ueda N. Maekawa M. Matsui T.S. Deguchi S. Takata T. Katahira J. et al.Inner nuclear membrane protein, sun1, is required for cytoskeletal force generation and focal adhesion maturation.Front Cell Dev Biol. 2022; 10885859Crossref PubMed Scopus (1) Google Scholar), 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. 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. Data availability statementDatasets 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 (Iglesias-Bartolome et al., 2018Iglesias-Bartolome R. Uchiyama A. Molinolo A.A. Abusleme L. Brooks S.R. Callejas-Valera J.L. et al.Transcriptional signature primes human oral mucosa for rapid wound healing.Sci Transl Med. 2018; 10eaap8798Crossref PubMed Scopus (83) Google Scholar; Uchiyama et al., 2019Uchiyama A. Nayak S. Graf R. Cross M. Hasneen K. Gutkind J.S. et al.SOX2 epidermal overexpression promotes cutaneous wound healing via activation of EGFR/MEK/ERK signaling mediated by EGFR ligands.J Invest Dermatol. 2019; 139: 1809-1820.e8Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar).Ethics statementAll procedures were approved by the National Institute of Arthritis and Musculoskeletal and Skin Diseases Animal Care and Use Committee. Data availability statementDatasets 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 (Iglesias-Bartolome et al., 2018Iglesias-Bartolome R. Uchiyama A. Molinolo A.A. Abusleme L. Brooks S.R. Callejas-Valera J.L. et al.Transcriptional signature primes human oral mucosa for rapid wound healing.Sci Transl Med. 2018; 10eaap8798Crossref PubMed Scopus (83) Google Scholar; Uchiyama et al., 2019Uchiyama A. Nayak S. Graf R. Cross M. Hasneen K. Gutkind J.S. et al.SOX2 epidermal overexpression promotes cutaneous wound healing via activation of EGFR/MEK/ERK signaling mediated by EGFR ligands.J Invest Dermatol. 2019; 139: 1809-1820.e8Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). 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 (Iglesias-Bartolome et al., 2018Iglesias-Bartolome R. Uchiyama A. Molinolo A.A. Abusleme L. Brooks S.R. Callejas-Valera J.L. et al.Transcriptional signature primes human oral mucosa for rapid wound healing.Sci Transl Med. 2018; 10eaap8798Crossref PubMed Scopus (83) Google Scholar; Uchiyama et al., 2019Uchiyama A. Nayak S. Graf R. Cross M. Hasneen K. Gutkind J.S. et al.SOX2 epidermal overexpression promotes cutaneous wound healing via activation of EGFR/MEK/ERK signaling mediated by EGFR ligands.J Invest Dermatol. 2019; 139: 1809-1820.e8Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Ethics statementAll procedures were approved by the National Institute of Arthritis and Musculoskeletal and Skin Diseases Animal Care and Use Committee. All procedures were approved by the National Institute of Arthritis and Musculoskeletal and Skin Diseases Animal Care and Use Committee. ORCIDsAndrew P. Sawaya: http://orcid.org/0000-0002-4675-3919Akihiko Uchiyama: http://orcid.org/0000-0003-0357-2808Emma Hope: http://orcid.org/0000-0002-9114-424XDeepti Bajpai: http://orcid.org/0000-0001-5701-8218Stephen Worrell: http://orcid.org/0000-0002-3605-7135Michael Cross: http://orcid.org/0000-0002-2446-9167Elia Beniash: http://orcid.org/0000-0001-9019-5160Lisa Jenkins: http://orcid.org/0000-0003-1245-1338Olivier Duverger: http://orcid.org/0000-0003-3200-1019Maria I. Morasso: http://orcid.org/0000-0002-9632-9593 Andrew P. Sawaya: http://orcid.org/0000-0002-4675-3919 Akihiko Uchiyama: http://orcid.org/0000-0003-0357-2808 Emma Hope: http://orcid.org/0000-0002-9114-424X Deepti Bajpai: http://orcid.org/0000-0001-5701-8218 Stephen Worrell: http://orcid.org/0000-0002-3605-7135 Michael Cross: http://orcid.org/0000-0002-2446-9167 Elia Beniash: http://orcid.org/0000-0001-9019-5160 Lisa Jenkins: http://orcid.org/0000-0003-1245-1338 Olivier Duverger: http://orcid.org/0000-0003-3200-1019 Maria I. Morasso: http://orcid.org/0000-0002-9632-9593 Conflict of InterestThe authors state no conflict of interest. The authors state no conflict of interest. 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. Conceptualization: APS, MIM; Data Curation: APS, AU, EH, DB, SW, MC, EB, LMJ, OD, MIM; Formal Analysis: APS, AU, EH, DB, SW, MC, EB, LMJ, OD, MIM; Funding Acquisition: MIM; Investigation: APS, AU, EH, DB, SW, MC, EB, LMJ, MIM; Methodology: APS, MIM; Project Administration: MIM; Resources: EB, LMJ, MIM; Supervision: MIM; Validation: APS, AU, EH, DB, SW, MC, EB, LMJ, OD, MIM; Writing - Original Draft Preparation: APS, DB, MIM; Writing - Review and Editing: APS, DB, MIM. Supplementary Material Download .pdf (.92 MB) Help with pdf files Supplementary DataeyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI4ZjNiYTk0ODkxNDkzMDA2YzI1MDIyZWU1YTg5NTcwOCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjY5OTM3ODA0fQ.lu6xExdaX-bWzd5NapDAjpTVE-i7s7SN0fwDl5k_vCbn0qesVL3YDzbkOw5uYSsicfUO9qoJP-1a6_0zDJu7-ZJVYtL8fbLSimn7u2k_q8P_YQkE9y7r8LM_uaF-Esw4wbu8iFHjtTe2iWB15uysmikaa-F3KGi3_3qBTM-wOpioYKQ7TMVpFZnu-pd8dTyewuJGc8CYjs93oWm7FS9DcIqzFiytGUo9T7WvKxamO-4bWFt9stE3n1M7KN7KAPSF60bq2j3nnfBAQ9cyLmKbJasaHHna2Taj7t8ZbiKTpyvket8tT_i5Q1liJCmT6UOn2cFy0oFTj0knORx6LOyGdg Download .mp4 (4.82 MB) Help with .mp4 files Supplementary Movie1 Download .pdf (.92 MB) Help with pdf files Supplementary DataeyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI4ZjNiYTk0ODkxNDkzMDA2YzI1MDIyZWU1YTg5NTcwOCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjY5OTM3ODA0fQ.lu6xExdaX-bWzd5NapDAjpTVE-i7s7SN0fwDl5k_vCbn0qesVL3YDzbkOw5uYSsicfUO9qoJP-1a6_0zDJu7-ZJVYtL8fbLSimn7u2k_q8P_YQkE9y7r8LM_uaF-Esw4wbu8iFHjtTe2iWB15uysmikaa-F3KGi3_3qBTM-wOpioYKQ7TMVpFZnu-pd8dTyewuJGc8CYjs93oWm7FS9DcIqzFiytGUo9T7WvKxamO-4bWFt9stE3n1M7KN7KAPSF60bq2j3nnfBAQ9cyLmKbJasaHHna2Taj7t8ZbiKTpyvket8tT_i5Q1liJCmT6UOn2cFy0oFTj0knORx6LOyGdg Download .mp4 (4.82 MB) Help with .mp4 files Supplementary Movie1