gli2 mice , in which the bovine k5 promoter drives the constitutive expression of mouse gli26 , develop bcc and bfh6,7 , which are both linked to deregulated hh signaling in humans7,8 .
gli2 mice show a reproducible pattern of lesions on the ear that successively involves hyper - keratosis ( flaking ) , thickening and hyperpigmentation ( supplemental fig .
mice were scored as positive for the onset of lesions upon the first sign of macroscopic hyperkeratosis in ear tissue .
histologically , the lesions present between p80 and p120 resemble bfh as described 7,8 . by p180 ,
larger , nodular , bcc - like tumors frequently occur deeper in the dermis ( supplemental fig .
induction of k17 , a gli target gene9 , is the main alteration in keratin expression prior to onset of lesions in gli2 epidermis ( supplemental fig .
gli2 and k17 mice6,10 were interbred so as to assess the impact of k17 loss on genesis of bfh - like tumors .
appearance and progression of hamartoma - like lesions were captured from p30 to p125 . at p80 ,
epithelial lesions are clearly less pronounced in gli2/k17 than in gli2 ear tissue ( fig .
in male gli2 and gli2/k17 mice the average onset of lesions is 652 days ( n=32 ) and 912 days ( n=31 ; p < 0.01 ) , respectively . in females
, onset is at 805 days ( gli2 ; n=22 ) vs. 1012 days ( gli2/k17 ; n=21 ; p < 0.01)(fig .
loss of k17 does not impact gli2 subcellular localization or hedgehog signaling ( supplemental fig .
therefore , the absence of k17 causes a delay in the inception of bfh - like skin tumors in gli2 mice .
histological anomalies common to hh pathway - activated mouse skin7 were scored in gli2 and gli2/k17 ear tissue ( supplementary fig .
3b ) , are prominent in gli2 ear but markedly reduced in gli2/k17 ear ( supplementary fig .
3c ) . overall tissue thickness and penetration of epithelial downgrowths are also reduced in gli2/k17 ear tissue ( supplementary fig .
k17 , k5 , and k14 are uniformly distributed in the lesional epithelium ( supplemental fig .
co - assembly of k5 and k17 in gli2 lesional epithelium is conveyed by their co - localization and co - immunoprecipitation ( supplemental fig .
the wound - inducible k6 , k6 and k16 , absent in intact epidermis , are induced in the upper layers of thickened gli2 epidermis , preferentially , but are markedly reduced in gli2/k17 skin ( supplemental fig .
reduced proliferation , rather than increased cell death , is a key contributor to delayed tumor onset in gli2/k17 skin .
relative to gli2 , indeed , the frequency of mitotically - active cells is depressed by > 3-fold in gli2/k17 ear epithelium ( fig .
g ) . in contrast , tunel - positive , apoptotic cells are restricted to the upper epidermis of lesional skin and show similar density in both genotypes ( fig .
inflammation has emerged as a driver of angiogenesis and tumor growth12 and coincides with k17 induction and loss of barrier function in several skin diseases13,14 .
immunoreactivity for markers of innate immune cells ( cd11b ) , t cells ( thy-1 ) , and phagocytes ( inos ) are enhanced in gli2 compared to gli2/k17 ear skin of p80 male mice ( fig .
2a ) , reflecting decreased angiogenesis . myeloperoxidase ( mpo ) enzymatic activity , inherent to neutrophils15 , is increased 17.4  0.5 fold in p80 male gli2 ear tissue but only 5.8  0.1 fold in gli2/k17 males ( data normalized to p80 female gli2 ear ; fig .
female gli2/k17 mice also show a reduced level of mpo activity at p80 , being at 0.75 fold of that seen in gli2 controls ( fig .
. skin barrier integrity , assessed via a whole - mount dye penetration assay16 , is intact as expected in p70 wildtype ear skin ( fig .
in contrast , a sizable portion of the ear is dye - permeable in p70 gli2 mice ( fig .
2c ) ; again , this readout is markedly decreased in gli2/k17 mice ( fig .
2d ) , mpo activity is 5.91.9 fold greater in male gli2 mice relative to females , substantiating the gender bias in this model .
mpo activity is lower in p40 male gli2/k17 mice ( 0.55  0.20 relative to female gli2 mice ; fig .
2d ) , mitotic activity is higher in gli2 vs. gli2/k17 epidermis at p40 ( 0.52  0.01 vs. 0.14  0.02 brdu labeled cells / mm of epidermis ) ( fig .
barrier integrity is mildy compromised in p40 gli2 ear skin , is again better preserved in gli2/k17 mice ( supplemental fig .
thus , the marked reductions in inflammation and hyperplasia that define gli2/k17 ear skin occur as early as p40 , ahead of progression to overt tumorigenesis in the gli2 model .
expression of inflammatory cytokines and chemokines was examined via qrt - pcr in ear tissue at p40 and p80 .
the findings are stratified according to specific classes of t - helper cytokines : th1 ( cellular immunity ; generally  pro - inflammatory  ) , th2 ( humoral immunity ;  anti - inflammatory  ) , and th17 ( anti - microbial immunity at epithelial barriers)17,18 . th1 and
absence of k17 in gli2 skin correlates with a marked reduction in th1- and th17-related markers and induction of th2-related markers ( table 1 ) , many of which are prominently expressed by skin keratinocytes themselves .
expression of il-1 , a keratinocyte mitogen20 , is ~10 fold higher in gli2 compared to gli2/k17 skin ( table 1 ) .
spp1 ( osteopontin ) , which acts to bias immune responses toward th121 , is reduced by ~15 fold and il-6 , associated with the acute phase response and upregulated in human bcc22 , is lowered ~17 fold in gli2/k17 skin ( table1 ) .
the matrix metalloproteases mmp3 , mmp9 and mmp13 , whose expression is enhanced in bcc23 , are downregulated in gli2/k17 ear tissue .
classical th2 type cytokines primarily secreted by t - cells , e.g. , il-4 and il-10 , are modestly altered whereas ccl24 and ccr4 , expressed by skin keratinocytes24 , are respectively ~9 and ~3 fold higher in gli2/k17 ear tissue ( table 1 ) .
il1 and cxcl5 expression is enhanced in the presence of k17 , while the th2 markers il20 and il4 are enhanced in its absence ( table 1 ) .
thus , the immunomodulatory influence of k17 is first manifested at an early stage in this model .
topical application of the phorbol ester , 12-o - tetradecanoylphorbol-13-acetate ( tpa)25 , to ear skin induces acute inflammation and epidermal proliferation ( fig .
3a , b ) , providing a tumor - free , dermatitis - like setting in which to assess the impact of k17 loss .
the latter curtails hyperplasia - driven epidermal thickening ( wt ear tissue : 34.12.3 m in tpa- vs. 10.40.3 m in vehicle - treated ; k17 ear tissue : 18.7 0.8 m in tpa- vs. 10.60.8 m in vehicle - treated ; fig .
markers related to compromised skin barrier function ( s100a826 , thymic stromal lymphoprotein ( tslp)14 , -defensin 27 ) show elevated mrna levels in tpa - treated wildtype skin ( fig .
a partial shift toward a th2-dominated cytokine profile is seen in tpa - treated k17 skin , though the magnitude of the changes is less than in gli2 skin .
the th1 chemokines cxcl5 , ccl3 and il-1 are reduced 2.4- , 3.0- and 1.7-fold , respectively , and the th2 cytokine il-20 is 7.1 fold higher in tpa - treated k17 skin relative to control ( table 2 ) .
skin keratinocytes from gli2 and gli2/k17 newborn mice were seeded for primary culture ( 48h ) , and treated with tpa ( 12h ) to assess whether key changes in cytokine / chemokine expression are keratinocyte - autonomous . under basal conditions , gli2 and
tpa induces a two - fold enhancement in gli2 keratinocyte proliferation by 12h , whereas gli2/k17 cells are unchanged ( supplemental fig .
levels of cxcl11 , cxcl5 , cxcl9 and cxcl10 mrnas , among others , are significantly lower in tpa - treated gli2/k17 keratinocytes ( fig .
these chemokines promote keratinocyte proliferation in skin tumors , and show a tight spatial correlation with k17 expression28,29 .
re - expression of k17 into tpa - treated gli2/k17 keratinocytes markedly elevates the levels of cxcl5 , cxcl9 , and cxcl11 mrnas , relative to mock - transfected cells ( fig .
k17 impacts the tpa - induced expression of select chemokines relevant to bcc pathogenesis in both adult epidermis in situ and isolated newborn keratinocytes in culture , suggesting that the mechanism(s ) involved are in part cell - autonomous .
several nf - kb target genes show a modest but consistent reduction in their expression in gli2/k17 relative to gli2 keratinocytes in tpa - treated cultures ( supplemental figure 5a ) .
this is consistent with the prominent role of nf-b in skin inflammatory conditions 30 and , in particular , with its impact on cxcl5 , cxcl9 , and cxcl11 expression3133 .
similar analyses of p80 whole ear skin tissue revealed no difference between the genotypes , likely reflecting the large complexity of these lesions in situ and the occurrence of secondary or compensatory changes ( supplemental figure 5 , b 
, k17 has been shown to promote anagen growth during hair follicle cycling34 and stimulates protein synthesis during tissue repair35 .
the phenotype reported here can not be correlated to obvious alterations in these roles , again as inferred from analyses of skin tissue sections ( data not shown ) or extracts ( supplemental fig . 5 , b 
k17 is ectopically expressed in numerous settings associated with robust inflammation including cutaneous wounds , various carcinomas , psoriasis , and virus - induced warts10 .
high levels of k17 expression correlate with a poor prognosis in breast36 and pancreatic37 cancers  whether this phenomenon is related to altered inflammatory signatures represents an issue of interest .
there exists a correlation between th1 hyperactivity and k17 expression in psoriatic plaques19 ; plaque resolution coincides with a shift to a th2 response and loss of k17 expression .
we posit that the presence of k17 in epidermis ( and related epithelia ) promotes hyperplasia in bcc - like tumors ( this study ) and likely in additional tumors and inflammatory disease settings in part through its ability to promote a specific type of inflammatory response .
normal contexts in which prominent k17 expression is not correlated to local inflammation ( e.g. , hair follicles ) may benefit from an immune - privileged status38 or reflect its regulation via post - translational modifications or interaction with other proteins34,35 . a role for k17 as an immunomodulator , whether direct or indirect , provides a novel way of conceiving how snps affecting k5 influence the risk of developing bcc 2 , and makes these keratins potentially attractive target for novel therapies aimed at curtailing conditions driven by or linked to chronic inflammation .
methods and any associated references are available in the online version of the paper at http://www.nature.com / naturegenetics/.
 basaloid skin tumors , including basal cell carcinoma ( bcc ) and basaloid follicular hamartoma ( bfh ) , are associated with aberrant hedgehog ( hh ) signaling1 and , in the case of bcc , an expanding set of genetic variants including keratin 5 ( k5)2 , an intermediate filament - forming protein . 
 we show that genetic ablation of keratin 17 ( k17 ) protein , which is induced in basaloid skin tumors3,4 and co - polymerizes with k5 in vivo5 , delays bfh tumor initiation and growth in mice with constitutive hh signaling in epidermis6,7 . 
 the delay is preceded by reduced inflammation and a polarization of inflammatory cytokines from a th1/th17- to a th2-dominated profile . 
 absence of k17 also attenuates hyperplasia and inflammation in a model of acute dermatitis . 
 re - expression of k17 in gli2tg k17/ keratinocytes induces select th1 chemokines with established roles in bcc . 
 our findings establish a novel immunomodulatory role for k17 in hh - driven basaloid skin tumors that could impact additional tumor settings , psoriasis , and wound repair . 
Main Text
METHODS
Supplementary Material