Under Pressure: A unique mechanoresponsive mechanism of body site-specific keratin regulation in palmoplantar epidermis

Under Pressure: A unique mechanoresponsive mechanism of body site-specific keratin regulation in palmoplantar epidermis | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Under Pressure: A unique mechanoresponsive mechanism of body site-specific keratin regulation in palmoplantar epidermis Sarah N. Steiner , Eric Horst , Mitre Athaiya , Craig N. Johnson , Joseph Y. Shen , Michelle L. Kerns , Geeta Mehta , Ramiro Iglesias-Bartolome , View ORCID Profile Pierre A. Coulombe doi: https://doi.org/10.1101/2025.09.15.676359 Sarah N. Steiner 1 Department of Cell & Developmental Biology, University of Michigan Medical School , Ann Arbor, MI Find this author on Google Scholar Find this author on PubMed Search for this author on this site Eric Horst 2 Department of Biomedical Engineering, University of Michigan Medical School , Ann Arbor, MI 5 Rogel Cancer Center, University of Michigan , Ann Arbor, MI Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mitre Athaiya 1 Department of Cell & Developmental Biology, University of Michigan Medical School , Ann Arbor, MI Find this author on Google Scholar Find this author on PubMed Search for this author on this site Craig N. Johnson 2 Department of Biomedical Engineering, University of Michigan Medical School , Ann Arbor, MI Find this author on Google Scholar Find this author on PubMed Search for this author on this site Joseph Y. Shen Find this author on Google Scholar Find this author on PubMed Search for this author on this site Michelle L. Kerns Find this author on Google Scholar Find this author on PubMed Search for this author on this site Geeta Mehta 2 Department of Biomedical Engineering, University of Michigan Medical School , Ann Arbor, MI 5 Rogel Cancer Center, University of Michigan , Ann Arbor, MI 6 Department of Materials Science and Engineering, University of Michigan , Ann Arbor, MI 7 Macromolecular Science and Engineering, University of Michigan , Ann Arbor, MI Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ramiro Iglesias-Bartolome 3 Laboratory for Cellular & Molecular Biology, National Cancer Institute , Bethesda, MD Find this author on Google Scholar Find this author on PubMed Search for this author on this site Pierre A. Coulombe 1 Department of Cell & Developmental Biology, University of Michigan Medical School , Ann Arbor, MI 4 Department of Dermatology, University of Michigan Medical School , Ann Arbor, MI 5 Rogel Cancer Center, University of Michigan , Ann Arbor, MI Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Pierre A. Coulombe For correspondence: coulombe{at}umich.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF SUMMARY Palmoplantar skin is structurally and molecularly distinct from other body sites. Notably, the type 1 keratin 9 ( KRT9 /K9) is exclusively expressed in palmoplantar epidermis. Mutations in KRT9 /K9 are causative for epidermolytic palmoplantar keratoderma (EPPK), a genetic disorder typified by palmoplantar keratoderma. Surprisingly little is known about the ontogeny, regulation and significance of Krt9 /K9. Here we characterize the regulation of Krt9 /K9 in postnatal palmoplantar epidermis and uncover a novel role of K9 in modulating YAP1 signaling. Expression of Krt9/ K9 rises dramatically post-birth, following a transient induction of the stress-related keratin 16 (Krt16/ K16 ). Krt9 null mice exhibit elevated K16 and aberrant nuclear-localized YAP1 by postnatal day 3. K9 interacts with 14-3-3σ to sequesters YAP1 in the cytoplasm, while EPPK-causing pathogenic variants impair these properties. Inhibition of YAP1 in vivo ameliorates palmoplantar keratoderma in Krt9 null mice. These findings provide novel insight into the adaptation of palmoplantar skin and suggest new therapeutic avenues for diseases featuring PPKs. Download figure Open in new tab HIGHLIGHTS Expression of Keratin 9 ( Krt9 /K9), a keratin specific to the thicker epidermis of palmoplantar skin, increases dramatically early after birth Onset of Krt9 /K9 expression follows, and is dependent on, K16, a stress-responsive keratin Krt9 null mice have aberrant nuclear YAP1 localization in differentiating keratinocytes of footpad skin Pharmacological or genetic inhibition of YAP1 rescues hyperkeratosis in Krt9 null mice Keratins play a crucial role in regulating differentiation and homeostasis in skin epithelia INTRODUCTION Specification of gene expression, cell fate, and ultimately function occurs in response to various cues and stressors in developing and mature organs and tissues. Skin epithelia, or epidermis, is an excellent example; it is the first line of defense against the external environment, providing protective functions including essential barrier function, water retention, immune surveillance, mechanical protection, and sensation ( 1 ) . Epidermis on the ventral aspect of the hands and feet is distinctive from other body sites on the histological, cellular and molecular levels and is known as palmoplantar skin. Owing to the substantial mechanical burden presented by locomotion, palmoplantar skin is characterized by specialized morphological and molecular adaptations ( 4 , 5 ) , among them the expression of keratins 9 (gene Krt9 ; protein K9) and 16 ( Krt16 /K16), in the differentiating layers of the epidermis ( 2 ) . The predominant cell type in the epidermis is keratinocytes, which express abundant keratin proteins, a diverse family of intermediate filament (IF)-forming proteins ( 3 ) . Keratins fall into one of two families of IFs—type I (acidic) and type II (basic-to-neutral) keratins ( 3 , 4 ) . Type I and type II keratins form obligate heterodimers, forming the essential building block of mature keratin IFs ( 4 ) . Keratin 9 (gene KRT9 ; protein K9) is a type I keratin that is exquisitely specific to the suprabasal (differentiating) layers of human and mouse palmoplantar epidermis ( 2 , 5 – 7 ) . K9’s presumptive type II binding partner is keratin 1 ( KRT1 /K1) ( 2 , 8 ) . Mutations in KRT9 /K9 cause epidermolytic palmoplantar keratoderma (EPPK), an autosomal dominant disorder characterized by fragility of suprabasal, differentiating keratinocytes, acanthosis, and hyperkeratosis in palmoplantar skin ( 9 , 10 ) , suggesting a loss of mechanical integrity and defects in tissue homeostasis ( 9 ) . Krt9 -/- mice recapitulate a clear EPPK-like phenotype ( 11 ) , developing macroscopic lesions in the footpad skin by 3.5 weeks of age, tissue fragility, and hyperkeratosis. There is, as yet, no successful clinical treatment for EPPK. Keratin 16 ( KRT16 /K16), also a type I keratin, is constitutively expressed in palmoplantar epidermis and epithelial appendages (e.g., hair, nail) but is not present in interfollicular epidermis unless the tissue is subject to injury or stress ( 12 ) . Notably, K16 can localize to the nucleus, although the nuclear function of K16 is not yet defined ( 13 , 14 ) . KRT16 /K16’s type II partner is keratin 6, of which there are three paralogs ( KRT6 /K6 A, B and C) ( 2 ) . Mutations in KRT16 /K16 result in pachyonychia congenita (PC), a condition that entails severe, but non-epidermolytic, palmoplantar keratoderma ( 15 ) . Several findings have illuminated a potential link between KRT16 /K16 and KRT9 /K9 in palmoplantar skin; KRT9 /K9 levels are decreased in PPK lesions of individuals with PC ( 16 ) and in Krt16 -/- mice ( 13 ) , which spontaneously develop PPK-like lesions. K16, on the other hand, is strongly upregulated in Krt9 -/- mice ( 11 ) . The relationship between K9 and K16 in palmoplantar skin remains unresolved. Here, we identify the novel role of Krt9 /K9 in regulating YAP1 signaling in the developing paw epidermis, suggesting a pathogenic mechanism relevant to PPK and related disorders. We show that Krt9 /K9 expression dramatically increases postnatally and follows a transient spike in KRT16 /K16 early after birth. In Krt9 -/- animals, aberrant nuclear-localized YAP1 in the suprabasal layers is coincident in this early postnatal period. We also show that K9 is sufficient to regulate the partitioning of YAP1 between the cytoplasm and nucleus in vitro , whereas EPPK-causing KRT9 variants fail to do so. Finally, we find that pharmacological and genetic inhibition of YAP1 rescues the EPPK-like lesions and reduces Krt16 /K16 expression in the Krt9 -/- mouse. These findings provide mechanistic insight into the specification and maintenance of palmoplantar keratinocyte identity, as well as potential clinical interventions for EPPK and other disease(s) manifesting with palmoplantar keratoderma. RESULTS Krt9 /K9 and Krt16 /K16 show unique expression patterns in early postnatal development WT mice were collected at embryonic day 18.5 (E18.5), postnatal day 0 (P0), P1, P3, and P10 of age. Whole paws were subjected to TMT-labelled quantitative mass spectrometry (TMT-MS). Upon examination of all intermediate filament species, only five displayed dynamic regulation between E18.5 and P10: keratin 16 ( Krt16 /K16), keratin 6 isoform b ( Krt6 /K6B), keratin 17 ( Krt17 /K17), keratin 2 ( Krt2 /K2), and keratin 9 ( Krt9 /K9) ( Fig. 1A ). Interestingly, four of these species stand out in their constitutive or exclusive expression in the palmoplantar skin, namely K16, K17, and the K6 paralogs, which are considered stress-inducible keratins in body epidermis, and K9, which exclusively marks the differentiating cells of the palmoplantar epidermis. Notably, these dynamics include a shift from K16/K6 dominance at P0-P1 to K9/K2 dominance at P3-P10. To further examine the dynamics of these unique keratins during this developmental interval, whole paws were collected from WT animals at E18.5, P0, P1, P2, P3, P10, and 3.5 weeks of age and subjected to Western blot (WB) and RT-qPCR analysis. The dynamic regulation of K16 was confirmed via WB ( Fig. 1B ). Levels of Krt9 significantly increase at P3, rising precipitously over development. Krt16 displays a dynamic regulation over developmental time, with relatively low levels at E18.5 and a dramatic rise between P0-P1 ( Suppl. Fig. 1A ). Bulk RNA-seq of whole paws collected from WT animals at P0 and P3 confirmed these patterns; Krt16 and Krt6b transcripts are enriched in P0 animals, while Krt2 and Krt9 transcripts are enriched in P3 samples ( Fig. 1C ). Transcripts associated with Krt16 / Krt6b expression in stress response ( 17 , 18 ) are also enriched in WT P0 samples; gene ontology (GO) terms such as keratinization, epidermal cell differentiation, and response to pain are associated with upregulated genes at P0 ( Fig. 1D ). WT P3 samples are enriched for GO terms that include intermediate filament organization and keratinocyte differentiation ( Fig. 1D ). Levels of Krt9 significantly increase at P3, rising dramatically over development. By contrast, Krt16 displays a dynamic regulation over developmental time, with relatively low levels at E18.5 and a significant but transient rise at P0-P1 ( Suppl. Fig. 1A ). From this, we hypothesized that Krt16 /K16 and Krt9 /K9 are responding to mechanical compression during the first use of the developing paw pad(s) during nursing and early locomotion. Download figure Open in new tab FIGURE 1: Krt9 /K9 and Krt16 /K16 are uniquely dynamic over developmental time and uncoupled from the specification of palmoplantar identity. A) TMT 10-plex labeled quantitative MS across WT development in the paw. N=2 animals per time point. Heat map represents Log 10 (Fold Change) relative to E18.5; see key. B) Western blot of K16 confirms dynamics over developmental time at E18.5, P0, P1, P3 and P10. Dotted line indicates the elimination of superfluous lanes. Krt16 -/- sample at right. N=2 animals per timepoint. Normalized relative to K14. One-way ANOVA. Data are represented as mean +/- SEM. C) Volcano plot representing bulk RNA sequencing of WT animals at P0 and P3. N=3 per timepoint/genotype. Significance cutoff at +/-1.5=Log 2 (Fold Change). D) GO terms enriched at P0 and P3, respectively. Palmoplantar keratins are responsive to compressive stress To assess whether KRT16 and KRT9 are responsive to mechanical compression in keratinocytes, we employed a compression bioreactor described in Novak et. al. ( 19 ) . Human A431 epidermal carcinoma cells were suspended in a 1% agarose/1mg/mL collagen type I hydrogel, approximating the Young’s modulus of viable epidermis of the plantar surface ( 20 , 21 ) . A431 cells were selected for their epithelial character and the absence of both KRT9 and KRT16 expression at baseline. Cells were subjected to 10 kilopascals (kPa) of static compression for 24 hours, in parallel to cells cultured under identical conditions but without the application of compressive force ( Fig. 2A , Suppl. Fig. 2A-A” ). Cells under compression displayed increased aspect ratios and cellular area ( Fig. 2B-B ’ ) relative to control cells. Compressed cells exhibited no change in proliferative activity as measured by Ki67 staining ( Suppl. Fig. 2B-B’ ) and otherwise remained viable in the setting of bioreactor culture. Key keratin transcripts normally expressed in the palmoplantar skin— KRT9 , KRT16 , and KRT17 —were all significantly upregulated in A431 cells post-compression ( Fig. 2C ), suggesting that their expression is responsive to mechanical stress. Download figure Open in new tab FIGURE 2: Compressive force stimulates palmoplantar-like keratin transcripts. A) Schematic of compression bioreactor, indicating components. Air is pumped into the underlying pressure chamber which deflects into the cell-laden interpenetrating hydrogel, deflecting the resistive membrane (black dome). The 3D cellular hydrogel component is held in place via a porous acrylic plug which also allows cell culture medium access from the top of this chamber. B) H&E images of control and compressed gels. Insets represent average areas of cells. Cells under compression exhibit a significant increase in cellular area and an increased aspect ratio (B’). Scale bar = 50µm. N=3; minimum of n=30 cells counted per condition. Data are represented as mean +/- SEM. Student t-test. C) RT-qPCR reveals upregulation of KRT9 , KRT16 , and KRT17 in A431 cells under compression. Log10 y-axis. Blue=control, red=compressed (10kPa, 24hrs). Fold change relative to 18S . Student t-test. Data are represented as mean +/- SEM. Krt9 -/- animals display aberrant YAP1 localization in the suprabasal layers of the palmoplantar epidermis We next set out to identify define the ontogeny of epidermal anomalies and mechanoresponsive pathways that may be altered in Krt9 -/- mice which, as related above, develop clear EPPK-like manifestations by 2.5-3 weeks of age (ref. 11 and Suppl. Fig 1 ). Krt9 -/- animals exhibit abnormally thickened palmoplantar epidermis as early as postnatal day 10 (P10) ( Suppl Fig 1C ); we therefore focused our attention on the first 10 days of life to detect the initial molecular alterations. YAP1 signaling, a mechanosensitive effector of terminal organ size of high significance ( 22 , 23 ) , has been implicated in the balance of proliferation and differentiation in keratinocytes of the interfollicular epidermis ( 24 , 25 ) . We have previously demonstrated that another keratin, namely K14, is essential for the regulation of YAP1 subcellular partitioning ( 26 ) . Consistent with previous reports in body epidermis ( 27 ) , a strong signal for YAP1 occurs in both the nucleus and cytoplasm in basal keratinocytes of the palmoplantar epidermis, and otherwise YAP1 occurs as a weaker and diffuse signal in the cytoplasm of suprabasal keratinocytes in WT skin via immunofluorescence (IF) ( Fig. 3A ” ). By contrast, Krt9 -/- mice exhibit aberrant nuclear-localized YAP1 in the suprabasal layers, specifically, as early as P3, as observed by IF ( Fig. 3A ’ ). By 3.5wks, when gross lesions are observable in Krt9 -/- mice, this phenotype has worsened ( FIG 3A” ). While overall levels of YAP1 are unaltered, we find that the ratio of phosphorylated YAP1 to total YAP1 is significantly attenuated in Krt9 -/- animals at P3 ( Fig. 3C ). Phosphorylated YAP1 is the form sequestered in the cytoplasm and therefore inactive ( 28 ) ; the reduction of phospho-YAP1 in Krt9 -/- animals correlates to the increase in its nuclear localization. Furthermore, the expression levels of the YAP1 binding protein 14-3-3σ are also significantly elevated in Krt9 -/- animals by P3 ( Fig. 3D ). We further confirmed that Krt16 /K16, which has previously been identified as elevated in the lesions of Krt9 -/- animals ( 11 ) are similar in WT and Krt9 -/- littermates at P0 but are elevated in Krt9 -/- animals, relative to WT, at P3 ( Fig. 3E ). Download figure Open in new tab FIGURE 3: Defects in YAP1 localization in Krt9 -/- palmoplantar epidermis. A) IF of K14/YAP1/DAPI in WT/ Krt9 -/- footpads. Krt9 -/- paw-pads exhibit aberrant nuclear YAP+ in the suprabasal layers compared to their WT littermates. P0 (A), P3 (A’), and 3.5wks (A”) of age shown. Red=K14, green=YAP1, blue=DAPI, scale bar = 50µm. Dashed line represents border between epidermis and dermis. B) Quantification of YAP1+ nuclei and distance from basal layer (A); N=3 biological replicates per age/genotype. Normalized relative to average WT epidermal thickness at each respective age point. Data represents individual values, bars indicating mean +/- SEM. One-way ANOVA. C) Quantification of western blots of phospho-YAP1, total YAP1, and 14-3-3σ in WT and Krt9 -/- P0/P3 animals. Normalized to Histone H3. N=3 animals/genotype per timepoint. Data are represented as mean +/- SEM. One-way ANOVA. D) Quantification of western blots for K16 in P0 and P3 in WT/ Krt9 -/- mice. K16 levels are elevated in Krt9 -/- animals as early as P3. N=3 animals/genotype per timepoint. Normalized relative to Histone H3. Data are represented as mean +/- SEM. One-way ANOVA. E) Bulk RNA-seq of WT/ Krt9 -/- animals at P0 and E’) P3 reveals minimal differences at P0, but notable changes in stress-related genes at P3. N=3 per genotype. Significance cutoff at +/- 1.5=Log 2 (Fold Change). K9 regulates YAP1 partitioning and activity via 14-3-3σ interaction In order to examine in vitro the disease relevance of K9’s newly found role in potentially impacting the subcellular partitioning of YAP1 in the epidermis in vivo , we generated two EPPK-relevant mutant K9 constructs: K9-GFP R163→Q (K9 R163Q), which is the most common EPPK-causative pathogenic variant in humans ( 9 , 29 ) , and K9-GFP C406→A (K9 C406A), representing the de-functionalization of the “stutter cysteine” residue ( Suppl. Fig. 3A-A’ ). Previously we utilized transfection-permissive HeLa cells to show that K14 mediates the cytoplasmic retention of YAP1 through, specifically, its stutter cysteine ( 26 ) . HeLa cells are particularly well-suited for these studies due to the strong nuclear YAP1 signal they exhibit when cultured on glass ( Fig. 4A ) and their robust expression of Cyr61 , a well-characterized YAP1 target gene ( 30 ) , allowing for the measurement of negative modulation of YAP1. We transfected the mutant K9 constructs, WT K9-GFP, WT EGFP-K14 (as a positive control), and GFP alone into HeLa cells and assessed the subcellular localization of YAP1 and the levels of Cyr61 in transfected cultures. In cells transfected with either WTK9-GFP or WT EGFP-K14, YAP1 showed a depletion from the nucleus and a reduction of nuclear-to-cytoplasmic fluorescence signal intensity, along with a concurrent reduction of YAP1 transcriptional activity as measured via RT-qPCR ( Fig. 4A-C ). By contrast, cells transfected with either K9 R163Q-GFP or K9 C406A-GFP did not deplete YAP1 from the nucleus and did not reduce levels of Cyr61 mRNA relative to baseline ( Fig. 4 A-C ). Overexpressed WT EGFP-K16 also did not result in YAP1 enrichment in the cytoplasm, despite sharing two key biochemical determinants with K14 and K9 ( Fig. 4 , Suppl. Fig. 3 ; see Discussion). The latter may account for the highly abnormal presence of YAP1 in the nucleus of suprabasal keratinocytes in the footpad skin of Krt9 -/- mouse despite the prevailing high levels of K16. Download figure Open in new tab FIGURE 4: WT K9 regulates the YAP/14-3-3σ complex, while EPPK-relevant mutations disrupt this role. A) Composite images of IF of YAP1 in HeLa cells transfected with WT and EPPK-mimic K9-GFP constructs. Blue= DAPI, purple =YAP1, green = GFP autofluorescence. Scale bar =20µm. B) Quantification of nuclear to cytoplasmic ratio of YAP1 in (A). DAPI was used to define nuclear boundaries; GFP autofluorescence of transfected constructs defined cytoplasmic boundaries. N=3 biological replicates, minimum 50 cells/condition/replicate. Data represents individual values, bars indicating mean +/- SEM. One-way ANOVA. C) Cyr61 transcription in transfected HeLa cells as measured by RT-qPCR. N=5 biological replicates. Student t-test. D) Proximity ligation assay (PLA) between GFP-tagged WT and EPPK-mimic K9 and endogenous 14-3-3σ in A431 cells. Blue=DAPI, green=GFP-tagged construct autofluorescence, red=PLA. Scale bar =10um. E) Quantification of PLA punctae/cell in (D). N=3 biological replicates; minimum 50 cells/condition/replicate. Data represents individual values, bars indicating mean +/- SEM. One-way ANOVA. Like most cytoplasmic partners that anchor YAP1 in the cytoplasm (e.g., catenins, 27 , 31 ), keratin proteins are unlikely to regulate YAP1 via direct binding and/or single-handedly. Instead, cytoplasmic partners such as catenins or K14 rely on 14-3-3 family isoforms, which bind both phosphorylated YAP1 and a phosphorylated residue on the cytoplasmic partner to form a ternary complex ( 26 , 27 ). Of particular interest is 14-3-3σ, a 14-3-3 family protein restricted to stratified epithelia ( 32 ). To test whether K9 interacts with 14-3-3σ, we performed co-immunoprecipitation (co-IP) in HeLa cells co-transfected with HA-tagged 14-3-3σ and WT K9-GFP ( Suppl. Fig. 3B ). Upon HA pulldown, we observed co-IP of both WT K9-GFP and of EGFP-K14, a known 14-3-3σ interactor ( 26 ) , indicating that these proteins indeed interact. To assess whether the interaction between K9 and 14-3-3σ holds in other cell culture systems and whether it is impacted by EPPK-causing pathogenic variants, we further performed proximity ligation assay (PLA) between transfected keratin constructs and endogenous 14-3-3σ in A431 cells ( Fig. 4D-E ). While an average of 7.7 PLA +/- 2.1 puncta per cell were observed in A431 cells expressing WT K9-GFP, a level comparable with cells expressing known 14-3-3σ interactor WT EGFP-K14, cells expressing either K9 R163Q (3.9 +/- 1.8 PLA puncta/cell) or K9 C406A (4.5 +/- 1.2 PLA puncta/cell) show significantly reduced instances of physical proximity with 14-3-3σ. These data show that the property of cytoplasmic sequestration of YAP1 is conserved in K14 and K9 but not in K16, and that the introduction of EPPK-causing pathogenic variants in K9 disrupts its ability to form a ternary complex with 14-3-3σ/YAP1 and negatively regulate the transcriptional activity of YAP1. Pharmacological inhibition of YAP1 rescues K9-dependent phenotypes We next hypothesized that inhibition of YAP1 may normalize the EPPK-like lesions in the footpad skin epidermis of Krt9 -/- mice. To that end, we first employed a pharmacological approach using verteporfin (VERT). VERT has been shown to inhibit YAP1 transcriptional activity by re-localizing it to the cytoplasm, though the precise mechanism underlying this effect is still unclear ( 33 – 35 ) . We tested a range of concentrations in HeLa cells in culture and found that treatment 2.5µM VERT effectively and significantly increases the phosphorylated-to-total YAP1 ratio, promoting its cytoplasmic localization ( Suppl. Fig. 4C-C’ ). The steady-state levels of YAP1 or 14-3-3σ proteins remained normal under these treatment conditions ( Suppl. Fig. 4C-C’ ). VERT treatment restored the cytoplasmic localization of YAP1 in cells transfected with K9 R163Q and K9 C406A ( Suppl. Fig. 4B-B” ). By contrast, no enhancement of YAP1 cytoplasmic sequestration was observed in cells transfected with WT EGFP-K14 or WT K9-GFP after VERT treatment (Suppl. Fig. 4B-B”). We next tested whether VERT treatment ameliorates the EPPK-like skin lesions characteristic of Krt9 -/- mice. WT and Krt9 -/- mice at 3.5 weeks of age—the age of first presentation of macroscopic lesions in the front paws in Krt9 -/- mice—were treated for 7 days with vehicle on the left paw and 3mg/mL verteporfin (ref. 36 , 37 ) on the right paw ( Fig. 5A ). Compared to vehicle treatment, VERT application resulted in a striking reduction (40 +/- 18.9%) of the thickness of the living layers of the epidermis in Krt9 -/- footpad skin ( Fig. 5B-C ). VERT treatment also reduced the total fraction of YAP1+ nuclei ( Fig. 5E-F ). Furthermore, YAP1+ nuclei occurred closer to the basement membrane, partially normalizing their distribution relative to WT ( Fig. 5F ). We further examined the levels of phospho-YAP1 in VERT-treated WT and Krt9 -/- paws and found that treatment with VERT improved the ratio of phospho-to-total YAP1 in the Krt9 -/- animals ( Fig. 5G ). To examine the mechanisms by which VERT may be normalizing the thickness of the Krt9 -/- palmoplantar epidermis, we performed RT-qPCR comparing WT and Krt9 -/- paws treated with vehicle control or VERT for 7 days and found that VERT-treated Krt9 -/- paws show a specific and significant reduction in Krt16 ( Fig. 5D ). This pattern was corroborated on the protein level via WB and IF ( Fig. 5D , Suppl. Fig. 5A, 5C ). Download figure Open in new tab FIGURE 5: In vivo treatment with verteporfin rescues YAP1 localization and normalizes epidermal thickness. A) Schematic of verteporfin (VERT) topical treatment. 3mg/mL VERT was applied for 7 days to the right paw; vehicle (VEH) was applied to the left. B) H&E of vehicle and verteporfin-treated footpads of WT and Krt9 -/- littermates. VERT selectively reduces the thickness of Krt9 -/- palmoplantar lesions. N= 6 animals per genotype (3 male, 3 female). Scale=100µm. C) Quantification of the thickness of the living layers of the epidermis in vehicle/verteporfin-treated palmoplantar epidermis. N=6 animals per genotype. Data represented as mean +/- SEM. One-way ANOVA. D) Krt16 transcript, measured via RT-qPCR, and K16 protein, measured via WB, in WT and Krt9 -/- paws treated with vehicle or VERT. RT-qPCR fold change relative to 18S . WB normalized relative to Histone H3. N=3 animals/genotype. Data represented as mean +/- SEM. One-way ANOVA. E) YAP1 staining, visualized via IF, in vehicle and verteporfin-treated WT and Krt9 -/- paws. Dashed line represents boundary between epidermis and dermis. Purple=K14, green=YAP1, blue=DAPI. Scale bar=50µm. N=6 animals/genotype. F) Quantification of YAP1+ nuclei in (E). Verteporfin treatment reduces the total fraction of YAP1+ nuclei (F) and their distribution relative to the basal layer (F’). Data represents individual values, bars indicating mean +/- SEM. One-way ANOVA. G) Quantification of WB of phospho-to-total YAP1 ratio in WT and Krt9 -/- animals treated with vehicle or VERT. N=3 per genotype. WB normalized relative to Histone H3. Data represented as mean +/- SEM. One-way ANOVA. YAP1 and the pro-differentiation transcription factor KLF4 ( 38 , 39 ) have been reported to interact and oppose one another in epidermal differentiation ( 24 ) . Given previous reports that Krt9 is a KLF4 target gene ( 40 ) , we further examined KLF4 localization in WT and Krt9 -/- mice treated with VERT. The lesional palmoplantar epidermis of vehicle-treated Krt9 -/- mice exhibited a significantly increased fraction of KLF4+ nuclei, relative to their WT littermates, while Krt9 -/- animals treated with VERT experienced a normalization of the fraction of KLF4+ nuclei ( Suppl. Fig. 5B-B’ ). Interestingly, WT animals treated with VERT experience an increase in Krt9 transcription ( Suppl. Fig. 5E ), suggesting a negative relationship between YAP1 activity and Krt9 /K9 expression. Blockage of YAP1 transcriptional activity ameliorates Krt9 -/- palmoplantar keratodermas As related above, various mechanisms have been proposed to account for VERT’s impact on YAP1 function. To determine whether the beneficial impact of VERT topical treatment on the footpad skin lesions of Krt9 -/- mice is due to reduced YAP1 transcriptional activity, we crossed Krt9 -/- mice with a recently described mouse line in which the expression of a synthetic TEAD inhibitor (TEADi) is placed under dual doxycycline and tamoxifen control in basal epidermal keratinocytes ( 24 ) . YAP1 binding to TEAD factors is essential for its transcriptional effects ( 41 ) ; hence, blocking YAP1 binding to TEAD factors should eliminate its transcriptional activity while leaving the localization, phosphorylation, and expression of endogenous YAP1 intact ( 24 ) . These mice carry a tetracycline-inducible synthetic TEADi gene (abbreviated tetO -TEADi), conditionally express reversible tetracycline inducible transactivator under the ROSA26 promoter (abbreviated ROSA26 lox-stop-lox [LSL]-rtTA mice) ( 42 ) , and carry a tamoxifen inducible Cre-recombinase under control of the cytokeratin 14 promoter (K14 CreERT ) ( 43 ) . In the resulting K14 CreERT ; ROSA26 LSL -rtTA ; tetO -TEADi animals (henceforth TEADi+), dual treatment with oral doxycycline and topically-applied tamoxifen blocks the transcriptional activity of YAP1 in the epidermis by preventing YAP1’s binding to its transcriptional co-activators (ref. 24 ; Suppl. Fig. 6A ). The resulting Krt9 -/- TEADi+ mice, along with key control animals (henceforth Krt9 +/+ TEADi+ and TEADi-, Krt9 -/- TEADi-) were treated with a combination of doxycycline chow and topically applied tamoxifen (TAM) or vehicle control (VEH) to the front paws ( Suppl. Fig. 6A-B ). Upon topical application of TAM, robust recombination was noted in animals carrying the TEADi cassette, as quantified via epidermal GFP fluorescence signal ( Suppl. Fig. 6C ). Moreover, the thickness of the palmoplantar lesions in Krt9 -/- TEADi+ animals were restored to the thickness of Krt9 +/+ TEADi+ and TEADi-littermates ( Fig. 6A-A ’ ). Krt9 -/- TEADi+ animals also exhibited significantly reduced staining for K16 ( Fig. 6B-B ’ ), corroborating the results from VERT-treated animals. Taken together, these data suggest that inhibition of YAP1 transcriptional activity is sufficient to ameliorate Krt9 -/- lesions and implicate Krt16 as a suprabasal transcriptional target of YAP1. Download figure Open in new tab FIGURE 6: Genetic inhibition of YAP1 ameliorates Krt9 -/- lesional presentation. A) Composite images of IF of tissue sections from Krt9 -/- TEADi+ mice and littermate controls. Krt9 -/- TEADi+ footpads exhibit decreased lesional thickness upon TAM/doxycycline administration. Red= E-cadherin, green=GFP, blue= DAPI. Scale bar=50µm. A’) Quantification living epidermal thickness in A). Minimum n=40 measurements/animal, N=4 animals/genotype/condition. Data represented as mean +/- SEM. One-way ANOVA analysis. B) IF of K16 (green) and DAPI (blue) in Krt9 -/- TEADi+ animals and littermate controls. Scale bar =50µm. B’) Quantification of K16 epidermal mean fluorescence intensity (MFI) in B). N=4 animals/genotype/condition. Data represented as mean +/- SEM. One-way ANOVA analysis. DISCUSSION Here, we report the postnatal mechanoresponsive regulation of K9 in palmoplantar epidermis and its role in maintaining homeostasis under the high mechanical demands of this specialized skin site. We further cement the dominant role of a cysteine residue located in the stutter motif of several type I keratins in mediating the cytoplasmic sequestration of YAP1 ( 26 ) , a transcription factor and terminal effector of the mechanoresponsive Hippo signaling pathway. We find that K9 interacts with the protein 14-3-3σ, another known determinant of YAP1 activity and localization in differentiating keratinocytes. Our findings establish a causative link between loss of proper YAP1 regulation and hyperkeratosis of the palmoplantar skin, suggesting a therapeutic target for EPPK and other disorders of palmoplantar epidermal differentiations. While EPPK resulting from pathogenic variants in KRT9 is an individually rare condition, the clinical presentation of palmoplantar keratoderma as a collective is not. Indeed, mutations in more than 50 genes result in disorders of palmoplantar epidermal differentiation and homeostasis ( 10 ) . Even patients with genetically determined fragility disorders potentially affecting the whole epidermis (e.g., epidermis bullosa simplex, EBS) experience relatively more severe dysfunction in palmoplantar skin ( 44 , 45 ) , as it represents the site of greatest mechanical stress. Accordingly, a more robust understanding of how homeostasis is established—and disrupted— in this specialized epidermal site stands to provide potential clinical insight for a wide variety of disorders. Previous studies on the development and specification of the palmoplantar skin have addressed distinct factors of KRT9 /K9 regulation, for instance identifying factors modulating palmoplantar identity in the epidermis and underlying mesenchyme. Ex vivo coculture of non-palmoplantar keratinocytes with palmoplantar fibroblasts highlighted the ability of mesenchymal cues to induce KRT9 expression ( 6 , 46 , 47 ) . Fibroblasts in the distal limb exhibit a unique HOX code, enabling the secretion of Wnt5a ( 48 ) , which is sufficient to induce KRT9 expression in non-palmoplantar human keratinocytes ( 6 , 49 ) . Furthermore, keratinocyte-intrinsic factors play a key part in modulating palmoplantar epidermal differentiation and expression of KRT9 /K9. For example, mutations in Wnt10A result in loss of KRT9 /K9 expression and the manifestation of PPK, caused in part by hypoactivation of the transcription factor KLF4 in the differentiating layers of the palmoplantar epidermis ( 40 ) . However, while the unique tissue architecture and identity of the palmoplantar surface, from the formation of fingerprints ( 50 ) and nail beds ( 51 ) to sweat glands in lieu of hair follicles ( 52 ) , is specified prior to birth, our findings establish that Krt9 /K9 expression is low relative to the high homeostatic levels prevailing in adult palmoplantar epidermis. Furthermore, Krt9’s presumptive type 2 partner Krt1 /K1 is robustly expressed by E16.5 in the developing mouse palmoplantar epidermis ( 53 ) , and the differentiation-, stress-, and developmental-dependent regulation of type I and type II keratin genes as pairs (e.g., Krt1/Krt10 ) is a prominent feature in the epidermis of most body sites ( 54 , 55 ) . The relatively low induction of Krt9 in ex vivo culture, along with its temporal uncoupling from the expression of its presumed type 2 partner, may bear a relationship to the unique postnatal mechanical demands placed on the palmoplantar epidermis. We find that, unlike Krt1 or Krt10 , Krt9 /K9 expression does not become robust until a few days postnatally, correlating with dramatic changes to the mechanical demand on the palmoplantar skin. Further, we have previously demonstrated that Krt16 -/- mice fail to robustly express Krt9 /K9, although the mechanism is still unclear ( 13 ) . Given the observed dynamics of Krt16 /K16 expression in perinatal mouse paw skin and the attenuation of Krt16 /K16 expression in Krt9 -/- animals treated with a YAP inhibitor, Krt16 /K16 likely provides an acute, YAP1-responsive signal that is necessary for mechanically sensitive induction of Krt9 /K9. There is, to our knowledge, no precedent for such a role on the part of a keratin protein. YAP1 signaling is a crucial regulator of mechanotransduction ( 22 ) , and its role in epidermal homeostasis is well-established ( 25 , 56 ) . Consistent with previous literature, we find that YAP1 occurs at low levels in the nucleus of differentiating keratinocytes ( 27 , 32 , 56 , 57 ) , while it prominently localizes to the nucleus in progenitor keratinocytes given a specific level of cellular crowding and/or integrin-mediated adhesion to the extracellular matrix within the basal layer ( 58 , 59 ) . Heterogeneity in the basal layer, particularly in areas of the body that may experience greater or lesser environmental stress (e.g., U.V., ref 60 , 61 ) may account for clusters of nuclear YAP1-positive basal cells interspersed with cells lacking nuclear YAP1. Later, in the spinous layers, phase separation and LATS1/2 phosphorylation govern the maintenance of inactivation of YAP1 ( 62 ) ; however, the mechanisms that maintain YAP1 in the cytoplasm during early differentiation were yet uncharacterized. Our previous work has identified keratins as crucial regulators of this phenomenon; we have proposed a “gating” effect in the basal layer, dependent on the occurrence of keratin species with conserved stutter cysteines ( 26 ) . Our original model, based on studies focused on K14, argues that specification of K14-containing keratin filaments facilitates the binding of 14-3-3σ and sequestration of YAP1 in the cytoplasm, after which keratinocyte differentiation can proceed. Per the model, at least two modifications are required on K14 to specify this role: formation of a homotypic disulfide bond involving the stutter cysteine, and a phosphorylation event that is presumably necessary to engage 14-3-3α. As keratinocytes differentiate, however, K14 ceases to be made, and K10 and, in the palmoplantar epidermis, K9, become the predominant type I keratin species featuring stutter cysteine residues. In the original model, we predicted the involvement of a “handoff” mechanism whereby cytoplasmic sequestration of YAP1 would necessitate, as differentiation proceeds, the involvement of a differentiation-specific keratin that features the stutter cysteine and is capable of binding 14-3-3. The findings reported here provide compelling evidence that K9 (and, based on sequence homology and other considerations ( 26 , likely K10 as well)) fulfils this role in the epidermis of palmoplantar skin. Importantly, KRT9 /K9 is the only keratin gene in which a pathogenic variant altering the stutter cysteine has been documented so far ( 63 ) as the C406→R variant is causative for a classical presentation of EPPK. The data we report here suggest that, in addition to potentially causing fragility of the keratin cytoskeleton ( 9 , 64 ) , EPPK-causative pathogenic variants in KRT9 crucially fail in the “handoff” of 14-3-3σ/YAP1 ternary complexes during differentiation. As inferred from our studies of Krt9 null mice, the presence of other type I keratin species that contain a stutter cysteine in the palmoplantar epidermis, such as Krt10 /K10 ( 26 ) , Krt16 /K16 (Suppl. Fig. 3A”), and Krt17 /K17 ( 26 ) , does not compensate for the loss of Krt9 /K9 with respect to YAP1 sequestration, suggesting the necessity of Krt9 /K9 in regulating YAP1 localization in the palmoplantar epidermis. Additionally, as we report here, YAP1 inhibition directly reduces the abnormally high Krt16 /K16 expression in the lesions of Krt9 -/- animals. Recent work has demonstrated that in the context of inflammation, YAP1 inhibition also ameliorates the severity of psoriatic lesions ( 65 ) , a disease in which KRT16 /K16 is highly expressed. Our data further suggest that while Krt16 /K16 is directly responsive to YAP1, it is not competent to modulate YAP1. Instead, Krt16 /K16, through direct or indirect mechanisms, promotes the expression of Krt9 /K9 and mediates the tissue’s adaptation to the experience of mechanical stress. What biochemical determinant(s) on K16 may be necessary for this function, as well as the molecular mechanisms involved and the role of regional specification, remain unknown. A revised and expanded model that incorporates recent findings on K15, which is expressed in a subset of progenitor keratinocytes in the basal layer and conspicuously lacks the stutter cysteine (see Redmond et al 66 ), and the current findings on the differentiation-specific K9, is presented in Figure 7 . The model proposes that expression of K15 at sufficiently high levels promotes the progenitor state in the relevant subset of keratinocytes; that the progressive buildup in K14 levels combined with its specification through posttranslational modification overcomes the influence of K15 and promotes the entry of progenitor keratinocytes into differentiation; and that either K9 (palmoplantar epidermis) or K10 (interfollicular epidermis) is required to maintain keratinocytes into the differentiation program ( Fig. 7 ). The sequential expression of specific type I keratins in surface epithelia such as epidermis therefore has a profound impact on the regulation of the balance between the progenitor and differentiating status of keratinocytes as well as their terminal differentiation. Importantly, keratins do not regulate proliferation or differentiation per se – instead, the suite of keratin species expressed in a keratinocyte or tissue at any given time promotes specific cellular states along the homeostatic continuum. While the model relies on the stutter cysteine and its profound impact on the partitioning of YAP1 between the nucleus and cytoplasm, open questions of high significance remain. These include the precise involvement of 14-3-3 (which are bivalent adaptor proteins; 31 , 67 ), the role of site-specific phosphorylation on keratins, and the mechanosensing mechanisms involved. Download figure Open in new tab FIGURE 7: Model of proposed YAP regulation in homeostasis and under stress in the epidermis. See Discussion for details. The dynamic regulation of K9 and YAP1 localization in the palmoplantar epidermis has significance beyond disease resulting from mutations in KRT9 itself. Some diseases that manifest with PPK correlate to nonfunctional or low levels of KRT9 /K9; most notably, a subset of individuals with pachyonychia congenita (PC, 16 ) have low levels of K9 in the lesional palmoplantar epidermis. Other palmoplantar epidermal differentiation disorders, such as those arising from mutations to desmosomes, interrupt the mechanical linkage of the keratin filament network to the periphery of the cell ( 68 , 69 ) . Additionally, individuals with WNT10A mutations ( 40 ) develop PPK due to defects in KLF4 and the subsequent failure to robustly express KRT9 /K9, and patients with mutations directly in KLF4 also develop PPK ( 70 ) . Given the findings described here, it is possible that diverse genetic causes of PPKs converge on the etiology of failed K9 expression, leading to dysregulated YAP1. A survey of KRT9 expression and YAP1 localization and activity in various palmoplantar epidermal differentiation disorders may allow pharmacological inhibition of YAP1 to be a promising intervention for a subset of PPKs. Taken together, our findings identify Krt9 /K9 as an important modulator of YAP1 signaling in the palmoplantar epidermis, dynamically expressed in response to mechanical stress. More broadly, K9 joins a growing list of keratin proteins that fundamentally regulate aspects of keratinocyte differentiation and epidermal homeostasis, especially in the unique context of mechanical stress. AUTHOR CONTRIBUTIONS This study was designed by SNS and PAC. Data was generated by SNS, EH, JYS, and MLK. Data analysis and interpretation were performed by SNS, PAC, EH, MA, CNJ, GM, RIB, and PAC. CJ, MA, EH, and GM provided technical and material support. Figures were designed by SNS, with input from EH. SNS and PAC prepared the manuscript, with input from all authors. DECLARATION OF INTEREST The authors declare no competing interests. METHODS Mouse models and treatments All mouse experiments involved animals in the C57/Bl6J background and were approved by the Institutional Animal Use and Care Committee of University of Michigan Medical School. The Krt9 −/− [ 11 ], Krt16 -/- [ 71 ], and TEADi [ 24 ] mouse strains were previously described. Animals were collected at E18.5, P0, P1, P3, P10, and 3.5wks of age. Equivalent numbers of male and female mice were collected. For topical treatment of verteporfin, verteporfin (Sigma-Aldrich, SML0534-25MG) was dissolved at 30mg/mL in 10μl DMSO; immediately prior to use, this stock was diluted to 3mg/mL ( 36 , 72 ) , and 10μl was applied to the ventral surface of the right paw; 10µL vehicle (DMSO) was applied to the left paw. The procedure was repeated once a day for seven days, and animals were collected 24 hours after the final topical application. For use of the TEADi mice, experimental and control animals were fed with 625mg/kg doxycycline chow (Inotiv, TD.01306) starting 24 hours before first treatment. Tamoxifen (Thermofisher Scientific, #J63509.ME) was dissolved at 2 mg/mL in DMSO and aliquoted in 10µL increments; immediately prior to use, one aliquot per mouse was thawed and applied to the ventral surface of the right paw, while vehicle (DMSO) was applied to the left. The procedure was repeated once a day for 3 days while animals were continually administered doxycycline; paws were collected 7 days after the first treatment (Suppl. Fig. 6). Tissue collection and cryosectioning Mice were euthanized and paw tissue was collected at specific time points as indicated. Paw samples were embedded in −40°C optimal cutting temperature compound (O.C.T, Sakura Finetek USA, #4583). Cryosectioning was performed at −20°C using a CRYOSTAR NX50 Cryostat (Thermo Scientific) and MX35 ultramicrotome blade (Epredia #3053835), and 5-μm thick cross-sections were cut and placed on positively charged microscope slides (VWR #48311–703) and stored at −40°C until further use. Cell lines and treatments HeLa and A431 cells were purchased from ATCC and routinely tested for mycoplasma using the MycoAlert® Mycoplasma Detection Kit (Lonza, LT07-118). Cells were cultured in DMEM medium (Gibco #11995–065) supplemented with 10% FBS and 0.01% Penicillin-Streptomycin (Gibco #15140–122). Cells were treated with 0-5 μM verteporfin (Sigma-Aldrich, SML0534-25MG) or 3µM VT107 (MedChemExpress #HY-134957) in DMSO for 24 hours post-transfection. Compression Device Use A431 cells were cultured in DMEM medium (Gibco #11995–065) supplemented with 10% FBS and 0.01% Penicillin-Streptomycin (Gibco #15140–122). Cells were collected from plates using 0.05% trypsin and pelleted before suspension at 10 million cells/mL in the interpenetrating hydrogel comprising of 1% agarose and 1mg/mL collagen type I (R&D Systems, #3443-100-01), as previously described [ 19 ]. The cell laden hydrogels were plated within the control or experimental wells of the compression bioreactor and allowed to gel for 15 minutes. Finally, the entire device was placed within the cell culture incubator (5% CO 2 , 37 °C) for 24 hours. Continuous air pressure was supplied by a syringe pump (New Era Infusion One, #NE-300) and monitored for a gauge pressure of 14-15kPa, as extrapolated from COMSOL models (see below). COMSOL Computational Modeling Hydrogel characteristics previously determined through SEM, porosimetery, and rheometry [ 19 ] were used as inputs for the COMSOL Multiphysics 5.5 computational analysis of applied solid mechanics and are provided in Suppl. Fig. 2. Linear elastic models were used to describe the application force and resultant third principle stress component. Linear elastic properties were assigned to both the hydrogel and membrane material characteristics and the application of pressure was described using an underlying boundary load to define the pressure application on the membrane (Suppl. Fig. 2). Indirect immunofluorescence For frozen tissue sections, slides stored at −40°C were taken to room temperature, dried for 10 min, fixed with 4% paraformaldehyde (PFA) (Electron Microscopy Sciences #15710) for 20 min at room temperature, followed by 3 washes of 5 min with 1× PBS. A circle was drawn around each tissue section with a hydrophobic barrier pen (CALIBIOCHEM #402176). Tissue sections were blocked with blocking buffer (2% normal donkey serum, 1% bovine serum albumin in 1× PBS) for 1 hour at room temperature. For cultured cell samples, cells were seeded on glass coverslips in 12-well plates and cultured overnight at 37°C and 5% CO2 to let cells attach. After treatment, cells were fixed with 4% PFA for 10 min, permeabilized with 0.5% Triton X-100 for 5 min, then blocked with blocking buffer (5% normal donkey serum in 1× PBS) for 1 h at room temperature. Unconjugated primary antibodies (cf. S1 Table ) were diluted in blocking buffer and applied overnight at 4°C. On the second day, samples were washed and incubated with fluorophore-conjugated secondary antibodies for 1 h at room temperature in dark. Samples were then stained with 1 μg/ml of DAPI (Milipore Sigma #268298), washed, mounted with coverslips via FluoroSave reagent (EMD Millipore #345789), and dried overnight. Both tissue sections and cultured cell samples were imaged using a Zeiss LSM800 confocal microscope (Zeiss, Germany). The antibodies used are listed in Table S1 . Laser intensity and detector gain were optimized for each fluor/channel. Quantification of YAP1 nuclear to cytoplasmic ratio Cells were stained for YAP1 and imaged as described above. Microscopy images were processed using ImageJ. Using the ImageJ freeform tool, the outer perimeter of the cell body was defined using the GFP channel in cells transfected with GFP-labeled constructs, and the YAP1 intensity mean value was quantified (whole-cell MFI). Next, the nucleus was defined using the DAPI channel and the YAP1 mean intensity value was calculated (nuclear MFI). Nuclear YAP1 MFI was subtracted from whole-cell MFI to generate cytoplasmic MFI. The nuclear-to-cytoplasmic ratio was calculated by dividing nuclear MFI by cytoplasmic MFI. All conditions were normalized relative to the nuclear-to-cytoplasmic ratio of cells transfected with pMAX-GFP. Protein Digestion and TMT labeling Tissue lysates were prepared using TriZOL reagent (Fisher Scientific #15596018) according to manufacturer’s instructions. Protein pellets were solubilized in 8M urea lysis buffer, as previously described ( 13 ) . Proteins were denatured by boiling with Laemmli buffer (Bio-Rad #1610747) supplemented with 10% β-mercaptoethanol (β-ME) at 95°C for 10 min. The samples were separated by SDS-PAGE and stained with SimplyBlue SafeStain (Thermo Fisher Scientific). The protein samples were processed and analyzed at the Mass Spectrometry Facility of the Department of Pathology at the University of Michigan. Gel slice(s) corresponding to between 40kDa and 100kDa were taken and destained with 30% methanol for 4 h. Upon reduction (10 mM DTT) and alklylation (65 mM 2-Chloroacetamide) of the cysteines, proteins were digested overnight with 500 ng of sequencing grade, modified trypsin (Promega) at 37° C. Peptides were extracted by incubating the gel with 150 µL of 50% acetonitrile/0.1% TFA for 30 min at room temperature. A second extraction with 150 µL of 100% acetonitrile/0.1% TFA was also performed. Both extracts were combined and dried using a vacufuge (Eppendorf). After reconstitution in 100 μL of 100 mM triethylammonium bicarbonate, the peptides were labeled with a TMT 10plex reagent (ThermoFisher; Cat #90110) following the manufacturer’s protocol. Labeling was quenched by the addition of 8 µL of 5% hydroxylamine. Finally, samples were combined and dried completely before desalting them with SepPak C18 cartridges. The final samples were reconstituted in 20 μL of a 0.1% formic acid/2% acetonitrile solution. Two μL of this solution to collect high-resolution LC-MS (liquid chromatography-mass spectrometry) data in both MS2 and multinotch MS3 modes. Liquid chromatography-mass spectrometry analysis (LC-multinotch MS3) To obtain superior quantitation accuracy, we employed multinotch-MS3 ( 73 ) which minimizes the reporter ion ratio distortion resulting from fragmentation of co-isolated peptides during MS analysis. Orbitrap Ascend Tribrid equipped with FAIMS source (Thermo Fisher Scientific) and Vanquish Neo UHPLC were used to acquire the data. Two µL of the sample was resolved on an Easy-Spray PepMap Neo column (75 µm i.d. x 50 cm; Thermo Scientific) at the flow-rate of 300 nL/min using 0.1% formic acid/acetonitrile gradient system (3-19% acetonitrile in 72 min; 19--29% acetonitrile in 28 min; 29-41% in 20 min, followed by a 10 min column wash using 95% acetonitrile and re-equilibration) and directly sprayed onto the mass spectrometer using EasySpray source (Thermo Fisher Scientific). FAIMS source was operated in standard resolution mode, with a nitrogen gas flow of 4.2 L/min, and inner and outer electrode temperature of 100 °C and dispersion voltage or −5000 V. Two compensation voltages (CVs) of −45 and −65 V, 1.5 seconds per CV, were employed to select ions that enter the mass spectrometer for MS1 scan and MS/MS cycles. Mass spectrometer was set to collect MS1 scan (Orbitrap; 400-1600 m/z; 120K resolution; AGC target of 100%; max IT in Auto) following which precursor ions with charge states of 2-6 were isolated by quadrupole mass filter at 0.7 m/z width and fragmented by collision induced dissociation in ion trap (NCE 30%; normalized AGC target of 100%; max IT 35 ms). For multinotch-MS3, top 10 precursors from each MS2 were fragmented by HCD followed by Orbitrap analysis (NCE 55; 45K resolution; normalized AGC target of 200%; max IT 200 ms, 100-500 m/z scan range). Transient transfection of EGFP-tagged keratin constructs The EGFP-K14WT and EGFP-K16WT constructs (pC3-EGFP vector backbone) have been described ( 74 , 75 ) , as have the WT K5 and WTK6-Myc constructs. pMAX-GFP was supplied by Lonza. The K9-GFP and K1-FLAG constructs were purchased from OriGene and GenScript, respectively (Origene Technologies #RG218091, GenScript #OHu19449). The K9 EPPK mutant constructs (K9 R163Q/K9 C406A) were generated by site-directed mutagenesis by the U-M Vector Core. Constructs were transiently transfected into HeLa using the SE Cell Line 4D-Nucleofector X Kit (Lonza #V4XC-1032) and Lonza 4D-nucleofector X unit, pulse code CN-114. A431 cells using the SF Cell Line 4D-Nucleofector X Kit (Lonza #V4XC-2032) and Lonza 4D-nucleofector X unit pulse code EQ-100. 1 μg of total plasmid was used to transfect every 0.4 million cells. Transfected cells were plated on coverslips for immunofluorescence assays or in 12-well plates for RNA or protein extraction and RT-qPCR, IP, or WB.. HeLa cells were allowed to rest for 24 h and then subjected to the desired analyses. A431 cells were allowed to rest for 48 hours before harvesting for analyses. Proximity ligation assay A431 cells were transfected using SF Cell Line 4D-Nucleofector kit (Lonza, V4XC-2032) and program EQ-100. 150,000 cells and 1 ug of total plasmid DNA were transfected per parameter. After transfection, cells were plated on #1.5 glass coverslips and cultured for 24 hours. After 24 hours, media was removed, and cells were rinsed with 1X PBS and fixed in 4% PFA/PBS for 10 minutes. Fixed cells were washed, permeabilized for 10 minutes in 0.1% Triton X-100 and blocked in 2.5% NDS/PBS overnight at 4C. Cells were then incubated for 1 hour at 37C with mouse anti-GFP antibody (Novus Biologicals, NB600-597SS) and rabbit anti-14-3-3σ antibody (Sigma-Aldrich, PLA0201). Following primary antibody incubation, cells were incubated with anti-rabbit PLUS and anti-goat MINUS DuoLink Probes (Sigma-Aldrich, DUO92002, DUO92006), and PLA signal was developed according to manufacturer protocol (Sigma-Aldrich, DUO92013). Coverslips were imaged with a 40X objective with a Zeiss LSM 800 confocal microscope. Laser intensity and detector gain were optimized for each fluor/channel. Images were taken as Z-stacks spanning 10µm at 1µm intervals. ImageJ was used to generate maximum intensity projection images, and the number of PLA punctae per cell was quantified using the ImageJ multi-point tool, using GFP autofluorescence to define the boundaries of the cell(s). Statistical analysis was performed using GraphPad Prism and Mann-Whitney tests. Quantitative real-time PCR analysis (qRT-PCR) For tissue, RNA was isolated using TriZOL (Fisher Scientific, #15596018) according to manufacturer’s protocol. For cells, RNA was isolated using Qiagen RNeasy mini kit (Qiagen #74104) following the manufacturer’s protocol. Total RNA from either tissue or cells was converted to complementary DNA (cDNA) using iScript cDNA Synthesis Kit (Bio-Rad #1708891). The cDNA obtained was subjected to qRT-PCR using the itaq Universal SYBR green kit (Bio-Rad, #1725122) and the CFX 96 Real-Time System (Bio-Rad). The PCR parameters for qRT-PCR were 95°C for 5 min, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. A “no cDNA template control” and a “melt curve” were included in every PCR run. The normalized expression value of the target gene was determined by first averaging the relative expression of the target gene for each cDNA sample (ΔCq = average Cq target gene—average Cq reference gene) and then normalizing the relative expression value of the experimental condition to the control condition (2-( ΔCq Experimental - ΔCq Control )). Primers used in qRT-PCR assays are listed in Table S2 . Western blotting and immunoprecipitation Whole cell lysates were prepared in NP-40 lysis buffer [0.5% NP-40, 150 mM NaCl, 20 mM Tris (pH 7.5), EDTA 1 mM, 1× cOmplete protease inhibitor cocktail solution (Millipore #11836170001), 1× Pierce phosphatase inhibitor cocktail solution (Thermo Scientific #A32957), milliQ water]. Briefly, cultured cells in plates were washed once with cold 1× PBS and transferred to ice. Lysis buffer was added, and cells were scraped off from the plate and rotated at 4°C for 1 h. Supernatants were collected after centrifugation (12,000 × g, 10 min) and total protein levels were measured using Pierce BCA Protein Assay Kits (Thermo Scientific #23227). Proteins were denatured by boiling with Laemmli buffer (Bio-Rad #1610747) containing 10% β-mercaptoethanol (β-ME) for 10 min at 95°C. SDS-PAGE electrophoresis was performed on 4% to 15% gradient gels (Bio-Rad #4561084) or 4% to 20% gradient gels (GenScript #M00656). Gel-bound proteins were transferred to nitrocellulose membranes (Bio-Rad #1620115) using a transblot turbo transfer system (Bio-Rad). Blots were blocked in 5% BSA in phosphate-buffered saline containing 0.1% Tween20 (PBST-T) for 1 h at room temperature and incubated overnight at 4°C with primary antibodies (cf. S1 Table ) diluted in the blocking buffer. Secondary antibodies (cf. S1 Table ) diluted in blocking buffer were applied for 1 h at room temperature. Blots were developed using SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Scientific #34580) or ECL Select Western Blotting Detection Reagent (Cytiva #RPN2235) and imaged using a FluorChem Q system (ProteinSimple). For immunoprecipitation (IP) of GFP and turbo-GFP tagged constructs, GFP-Trap and turboGFP-Trap Magnetic Agarose (Proteintech, #tbtma-20, #gtma-100) were used, and 25 μl of beads were used. HeLa and A431 cells were lysed and diluted according to manufacturer’s direction. Whole cell lysates (600 μg of total protein) were incubated with beads at 4°C for 1hr. The eluted IP samples were denatured and subjected to western blotting. Bulk RNA sequencing Krt9 -/- and WT littermates were collected at postnatal day 0 and 3 (N= 3 mice for each group), and RNA was isolated and purified from whole paws using TriZOL reagent (Fisher Scientific, #1559601) using the manufacturer’s instructions. RNA samples were transferred to an external contractor (Novogene) for RNA quality control, RNA ribo-depletion library preparation, and next-generation sequencing (NovaSeq S4 300 cycle). The contractor supplied FASTQ files were mapped to the GRCm39.vm30 reference using STAR version 2.7.10a and the ENCODE standard options from the version documentation ( 76 ) . The reference matching GTF file was used with featureCounts from the Bioconductor R package Rsubread to create the counts matrix. The edgeR Bioconductor package was used to filter low expressing genes using the “filterByExpr” function, calculate log counts per million, and normalization factors using the weighted trimmed mean of M-values (TMM) method. The Limma Biocoductor package, with the precision weights, “voom” approach was used to perform linear models ( 77 ) . Differential gene expression was determined using custom thresholds as described for each analysis in the main text and in figure legends. SUPPLEMENTARY FIGURE LEGENDS Supplemental Figure 1: Molecular and histological defects in Krt9 -/- animals over developmental time. A) RT-qPCR data of Krt9 and Krt16 transcripts over WT developmental time. N=2 animals per timepoint. B) Quantification of western blot data tracking molecular defects in Krt9 -/- animals are evident at P3 and absent at P0. B) K14 and B’) KLF4, markers of basal and differentiating identity, respectively, are elevated. Normalized relative to Histone H3. N=3 animals/genotype/age. C) H&E staining of WT/ Krt9 -/- littermates from P5-3.5 weeks of age reveals histological phenotype as early as P10. N=2 per genotype/timepoint. Quantification of epidermal thickness in C) is presented in C’). Student t-test analysis. Supplemental Figure 2: Compressive force COMSOL modeling and cell viability. A) Mesh construction on COMSOL model of the hydrogel (1 cm height by 6 mm radius) and deflectable membrane (1 mm height by 6mm radius). COMSOL was used to calculate A’) extrapolation of input air pressure versus modeled internal compressive force using 3 methodologies on 1% agarose/1 mg/mL collagen 1 gel. Simple pressure was determined to be the best model; force distribution throughout the gel using the simple pressure model is shown in A”). B) IF of control and compressed A431 cells exhibiting normal proliferation, quantified in B’). Purple =β-actin, green=Ki67, blue=DAPI. Scale bar= 50µm. N=3. Data represented as mean +/- SEM. Student t-test analysis. Supplemental Figure 3: Characterization of novel EPPK-mimic KRT9 constructs and biochemical phenotypes. A) Schematic of EPPK-causative mutations on K9 and mutagenized residues; A’) the R163 residue frequently mutated in KRT9 -driven EPPK is conserved across K16 and K14, as is the A”) stutter cysteine residue. B) Western blot demonstrating co-IP of GFP-tagged keratins with HA-tagged 14-3-3σ. IN=input fraction, 10ug total protein loaded. IP=precipitated fraction, 600ug total protein loaded. kDa= kiloDaltons (approx. molecular weight). C) Quantification of western blots for YAP1 and 14-3-3σ of HeLas transfected with WT and EPPK-mimic KRT9 constructs. N=2 biological replicates. Data represented as mean +/- SEM. One-way ANOVA analysis. D) Western blot quantification of endogenous 14-3-3σ in A431 cells transfected with WT and EPPK-mimic KRT9 constructs. N=2 biological replicates. One-way ANOVA analysis. E) Quantification via R code of punctate GFP aggregates in HeLa cells transfected with WT and EPPK-mimic KRT9 constructs. K9 R163Q selectively forms dense, punctate aggregates. N=3, minimum 50 cells/condition/replicate. Data represented as mean +/- SEM. One-way ANOVA analysis. F) Morphological characterization of filaments in HeLas transfected with GFP-tagged keratin constructs. EPPK-relevant mutations disrupt normal filament morphology in transfected HeLa cells. N=3, minimum 50 cells/condition/replicate. Data represented as mean +/- SEM. Two-way ANOVA analysis. Supplemental Figure 4: Verteporfin re-localizes YAP1 to the cytoplasm and reduces its transcriptional activity in vitro . A) IF of YAP1 in HeLas treated with concentrations of verteporfin ranging from 0-5 µM. Blue=DAPI, green=YAP1. Scale bar=10µm. A’) Quantification of (A). Average of YAP1 nuclear/cytoplasmic mean intensity, N=2. One-way ANOVA analysis. B) IF of HeLa cells transfected with GFP-tagged keratin constructs. B’) EPPK-relevant K9 mutants treated with 2.5µM verteporfin exhibit cytoplasmically localized YAP1. Blue=DAPI, purple=YAP1, green=GFP-tagged construct autofluorescence. Scale bar =20um. B”) Quantification of YAP1 fluorescence nuclear to cytoplasmic ratio in (B). N=3, 50 cells/condition/replicate. Dots represent individual cell value(s), bars representing mean +/- SEM. One-way ANOVA analysis. C) WB of 14-3-3σ, YAP1, and Histone H3 in verteporfin-treated HeLas. C’) Quantification of YAP1, 14-3-3σ and phosphorylated-to-to-total YAP1 ratio in verteporfin and VT107-treated HeLas. Normalized relative to Histone H3. N=3. Data represented as mean +/- SEM. One-way ANOVA. Supplemental Figure 5: Verteporfin treatment normalizes additional molecular phenotypes in the Krt9 -/- mouse. A) IF of K16 in WT and Krt9 -/- animals treated with VERT. Red=K14, green=K16, blue=DAPI. Scale bar= 50μm. N=2 animals/condition. B) IF of KLF4 in WT/ Krt9 -/- animals treated with verteporfin. Red=K14, green=KLF4, blue=DAPI. Scale bar=50μm. Quantification of total fraction of KLF4+ nuclei in B’). N=3 animals/condition. Data represented as mean +/- SEM. One-way ANOVA. C) Western blot of K16 and Histone H3 in WT/ Krt9 -/- animals treated with verteporfin. N=3 animals/condition. Data represented as mean +/- SEM. One-way ANOVA. D) RT-qPCR of Krt9 from WT and Krt9 -/- paws treated with verteporfin. Krt9 expression is enhanced in VERT-treated WT mice. N=3 animals/condition. Data represented as mean +/- SEM. One-way ANOVA. E) Quantification of western blots of 14-3-3σ in WT/ Krt9 -/- animals treated with verteporfin. N=3 animals/condition. Data represented as mean +/- SEM. One-way ANOVA. Supplemental Figure 6: Generation and validation of Krt9 -/- TEADi+ mouse A) Genetic schematic of the Krt9 -/- TEADi+ mouse. B) Combination doxycycline/tamoxifen treatment schematic of Krt9 -/- TEADi+ animals and their control counterparts ( Krt9 +/+ TEADi+, Krt9 -/- TEADi-, Krt9 +/+ TEADi-, and TEADi+ animals not administered DOX/TAM). C) Epidermal GFP autofluorescence measured across all treatment and genotype conditions, demonstrating effective recombination only in TEADi+ samples administered both DOX and TAM. Data represented as mean +/- SEM. N=4 animals/condition. One-way ANOVA. TABLE TITLES AND LEGENDS View this table: View inline View popup Download powerpoint Table S1: Primary and secondary antibodies used in these studies. View this table: View inline View popup Download powerpoint Table S2: Genotyping and RT-qPCR primers used in these studies. All primers were purchased from IDT. Genotyping primers were designed based on previous publications (ref. 24 , 11 , 42 , 43 ). RT-qPCR primers were optimized using the IDT PrimerQuest tool. ACKNOWLEDGMENTS This research was supported in part by grants from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) to P.A.C. [R01 AR079418] and S.N.S. [F31 AR083249]. Research reported in this publication was supported by the National Cancer Institutes of Health under award number P30CA046592 given the use of Rogel Cancer Center Shared Resource(s) including the Tissue and Molecular Pathology Shared Resource and Proteomics Resource Facility. We also thank the Vector Core Facility at the University of Michigan and acknowledge Novogene for their assistance with the bulk RNA sequencing. 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