Defining a Region of the Human Keratin 6a Gene That ConfersInducible Expression in Stratified Epithelia of Transgenic Mice*

(Received for publication, October 15, 1996, and in revised form, January 28, 1997)

Kenzo Takahashi‡ and Pierre A. Coulombe§

From the Departments of Biological Chemistry and Dermatology, The Johns Hopkins University School of Medicine,Baltimore, Maryland 21205

Injury to the epidermis and other stratified epitheliatriggers a repair response involving the rapid inductionof several genes, including keratin 6 (K6). The signalingpathways and mechanisms presiding over this induc-tion in keratinocytes at the wound edge remain to bedefined. We reported previously that of the multiplegenes encoding K6 isoforms in human, K6a is dominantin skin epithelia (Takahashi, K., Paladini, R., Coulombe,P. A. (1995) J. Biol. Chem. 270, 18581–18592). Using bac-terial LacZ as a reporter gene in transgenic mice, weshow that the proximal 5.2 kilobases of 5*-upstream se-quence from the K6a gene fails to direct sustained ex-pression in any adult tissue, including those where K6 isconstitutively expressed (e.g. hair follicle, nail, oral mu-cosa, tongue, esophagus, forestomach). In contrast, theproximal 960 base pairs of 5*-upstream sequence sufficeto mediate an induction of b-galactosidase expression ina near-correct spatial and temporal fashion after injuryto epidermis and other stratified epithelia. Transgeneexpression also occurs following topical application ofphorbol esters, all-trans-retinoic acid, or 2–4-dinitro-1-fluorobenzene, all known to induce K6 expression inskin. Our data show that critical regulatory sequencesfor this inducibility are located between 2960 and 2550bp in the 5*-upstream sequence of K6a and that theiractivity is influenced by enhancer element(s) locatedbetween 22500 and 25200 base pairs. These findingshave important implications for the control of gene ex-pression after injury to stratified epithelia.

Injury to skin triggers a repair response aimed at restoringepithelial continuity and barrier function. The activity of sev-eral genes encoding intracellular, cell surface, and secretedproteins is rapidly modulated in the epithelial and mesenchy-mal cells involved in this response (1). Keratins 6, 16, and 17,the gap junction protein connexin 26, the receptor for theurokinase-type plasminogen activator, and various proteasesare induced in wound edge keratinocytes within hours afterinjury, and their subsequent accumulation correlates with ma-jor changes in keratinocyte cytoarchitecture that precede theonset of migration toward the wound site (2). While the signif-icance of these changes remains to be elucidated, the study of

the regulation of the corresponding genes offers an opportunityto decipher the molecular mechanisms underlying the onset ofthe wound repair response in stratified epithelia. Indeed, eventhough the levels of several potent growth factors are greatlyelevated in the wound site early after injury to the skin (re-viewed in Ref. 3), those playing a critical role in this vitalhomeostatic response remain to be identified.

We recently cloned several human genes and cDNAs pre-dicted to encode highly related keratin 6 (K6) isoforms (4). K6(56 kDa) is a type II keratin that belongs to the superfamily ofintermediate filament proteins and is ususally co-expressedwith one or two type I keratins, K16 and K17 (5, 6). The K6isoforms show a complex pattern of expression in epithelia,with constitutive and inducible components (7). They are nor-mally found in the outer root sheath (ORS)1 of hair follicles, inglandular tissues, in tongue, gingiva and oral mucosa, esoph-agus, forestomach, and certain reproductive tract epithelia (e.g.Ref. 8). With the exception of palm and sole, K6 is not expressedin normal interfollicular epidermis (5, 7, 8). The K6 isoformsare better known for their much enhanced expression duringhyperproliferation and abnormal differentiation in stratifiedepithelia (4, 9, 10). Thus, K6 and K16 are induced in woundedge keratinocytes as early as 4–6 h after injury to human skinand disappear after closure (11, 12). K6 expression is inducedas well in a variety of diseases affecting complex epithelia, suchas infections, squamous metaplasia, carcinoma, and chronichyperproliferative disorders, including psoriasis (9, 10). Inthese conditions, K6 expression may be very abundant, but isusually restricted to the suprabasal compartment of the epi-thelium (10). In mouse skin, K6 expression is induced aftertopical application of a variety of chemicals (e.g. phorbol esters,retinoic acid; see Ref. 13). K6 induction also occurs in primarycultures of mitotically active keratinocytes from epidermis,esophagus, trachea, and cornea (7, 14). Understanding theregulation of K6 gene expression is thus of great interest atvarious levels, one being the control of gene expression incontexts such as wound repair, psoriasis, and carcinoma. Usinga transgenic mouse approach, we report here on the identifica-tion of a segment of 59-upstream sequence in the human K6agene that is both necessary and sufficient for the inducibleexpression of an heterologous reporter gene in adult mouseepithelia.

EXPERIMENTAL PROCEDURES

DNA Constructs and Production of Transgenic Mice—Our startingtemplate was the human K6a gene, the dominant K6 isoform in hairfollicle outer root sheath, foot sole epidermis, and skin squamous car-cinoma samples (4). Segments containing 5.2 kb (SmaI-NcoI), 2.56 kb(HindIII-NcoI), 0.96 kb (EcoRI-NcoI), and 0.55 kb (SacI-NcoI) of 59-

* This work was supported by National Institutes of Health GrantAR42047. The costs of publication of this article were defrayed in partby the payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

‡ Fellow supported by the Dermatology Foundation.§ Recipient of a Junior Faculty Research Award from the American

Cancer Society. To whom correspondence should be addressed: Dept. ofBiological Chemistry, Johns Hopkins University School of Medicine,725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-614-0510; Fax: 410-955-5759; E-mail: pacoulomb@welchlink.welch.jhu.edu.

1 The abbreviations used are: ORS, outer root sheath; kb, kilobasepair(s); bp, base pair(s); X-gal, 5-bromo-4-chloro-3-indolyl b-D-galacto-pyranoside; PMA, phorbol 12-myristate 13-acetate; DNFB, 2–4-dinitro-1-fluorobenzene; RA, all-trans-retinoic acid.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 18, Issue of May 2, pp. 11979–11985, 1997© 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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upstream sequence from the translation initiation codon were isolatedfrom the human K6a gene (GenBank accession numbers L42575–L42583; see Ref. 4) by restriction digestion and subcloned into a LacZexpression cassette. We used a LacZ coding sequence (plasmid pCH110)modified to contain a nuclear localization sequence at its 59-coding endin addition to the SV40 poly(A) sequence at its 39 end (15). The fourtransgene constructs devised are as follows: KT1, [5.20-kb hK6a 59-upstream sequence]-LacZ; KT2: [2.55-kb hK6a 59-upstream sequence]-LacZ; KT3, [0.96-kb hK6a 59-upstream sequence]-LacZ; KT4, [0.55-kbhK6a 59-upstream sequence]-LacZ.

Transgenic mice were produced by pronuclear injection of DNA con-structs in single cell C57B6/BalbC3 embryos (16). Founders were iden-tified by Southern blotting of genomic DNAs using a probe to the codingportion of LacZ. Transgenic lines were established by matings in theC57B6/BalbC3 mixed background (Jackson Laboratories). Double-transgenic mice were produced by mating the previously describedPC5-7-K16 transgenic mice, which contain 8–10 copies of the full-length human K16 gene (17), with KT2-2m transgenic mice (Table I).

b-Galactosidase Histochemistry and Enzymatic Assays and TissueSection Immunostainings—For b-galactosidase histochemistry in situ,adult mouse tissues were prefixed in 1% formaldehyde, 0.2% glutaral-dehyde (60 min), washed in phosphate-buffered saline, incubated over-night at 30 °C in a solution containing 1 mg/ml X-gal, 100 mM sodiumphosphate buffer, pH 7.3, 1.3 mM MgCl2, 3 mM K3Fe(CN)6, and 3 mM

K4Fe(CN)6 (18), post-fixed in Bouin’s, and paraffin-embedded. 5-mmsections were counter-stained with eosin. For immunohistochemistry,Bouin’s-fixed tissues were paraffin-embedded, and 5-mm sections werereacted with antisera directed against mouse K6 (19) or anti-b-galac-tosidase (Promega, Madison, WI). Bound primary antibodies were re-vealed by a peroxidase-based reaction as recommended (Kirkegaardand Perry Laboratories, Gaithersburg, MD). For biochemical analysis,adult mouse skin tissue extracts were prepared by homogenization inb-galactosidase reporter lysis buffer, and post-centrifugation superna-tants were used for the detection of b-galactosidase enzymatic activityfollowing the manufacturer’s instructions (Promega).

Experimental Injury and Chemical Treatment of Mouse Tissues—Allstudies involving animals were reviewed by the Johns Hopkins Univer-sity Animal Use and Care Committee. For studies involving skin, adultmice (3–6 months old) were anesthetized with avertin and their backsepilated with Nair cream. For injury, the surgical area was disinfected,and full thickness skin wounds were made with a 4-mm punch (Acu-

Punch; Acuderm Inc., Ft. Lauderdale, FL). For studies involving otherstratified epithelia, adult mice were anesthetized, and short superficialincisions were made with a sterile scalpel to either foot pad epidermis,cornea, oral mucosa, or tongue. Tissues were harvested after 24 h andprocessed for b-galactosidase histochemistry as described above. Forstudies involving chemical treatment of skin, solutions of PMA (phor-bol-12-myristate-13-acetate, 150 ml of a 50 mM stock in acetone; Sigma)and all-trans-retinoic acid (150 ml of a 100 mg/ml stock in ethanol;Sigma) were applied topically on Nair-epilated skin every 3rd day forthree times. To induce a delayed-type skin hypersensitivity reaction,mice were sensitized with an application of 25 ml of 0.25% 2–4-dinitro-1-fluorobenzene (DNFB) at the base of the tail and challenged 5 dayslater by application of 10 ml of the same solution onto the dorsal neckarea as described (20). The mice were sacrificed and the skin processedfor b-galactosidase histochemistry on the next day. Skin papillomaswere induced using the two-step chemical carcinogenesis procedure(21), involving initiation with 7,12-dimethylbenz[a]anthracene and pro-motion with PMA for 11 weeks. Papillomas were harvested 1 monthafter cessation of treatment and processed for analysis. In all experi-ments involving chemical inducers, controls consisted in application ofthe vehicle only.

RESULTS

The Human K6a 59-Upstream Sequence Fails to Direct Sus-tained Expression in Adult Mouse Tissues—Table I lists thetransgenic lines produced for each of the constructs and reportson transgene expression assessed by b-galactosidase histo-chemistry in situ. None of the constructs, including KT1 (5.2 kbof K6a 59-upstream sequence), shows consistent expression inthe ear, trunk, tail, or paw skin of adult transgenic mice (Fig.1A). In KT1, KT2, and KT3 lines, occasional ORS keratinocytesdisplay b-galactosidase activity in a subset of hair follicles(Table I; Fig. 1A). Generally, fewer that three to five folliclesshow sporadic X-gal staining in the ORS in a typical section(1–2 cm wide) of adult skin tissue (Fig. 1B). In contrast, endog-enous K6 is easily detected in mouse hair follicles (Fig. 1C).These findings are supported by immunostainings using anti-bodies against the b-galactosidase protein, by Northern blot-

TABLE I[K6a 59]-LacZ transgene expression in transgenic mouse skin

Promoterregion TG line TG copy

number

Intact trunk skina Wounded tissueb

Other tissuesEpidermis Hair

follicle Skin Oralmucosa

0.5 kb KT4–1p 2 2 2 2 2KT4–2p 4 2 6c 6 2KT4–3p 4 2 2 2 2KT4–1m 4 2 2 2 2 Retinad

KT4–2m 1 2 2 6 2KT4–3m 2 2 2 2 2

1.0 kb KT3–2p 3 2 6c 1 2 Nail bed,d lungd

KT3–1m 1 2 2 6 6 Retinad

KT3–2m 5 2 6c 11 11 Cornea,d esoph,d kid,d trach,d pericardd

KT3–3m 1 2 6c 11 1KT3–4m 4 2 6c 11 11 Tongue,d esoph,d retinad

2.5 kb KT2–1p 2 6c 6c 1 11 Tongued

KT2–2m 4 2 6c 11 11 Retinad

5.2 kb KT1–1p 1 2 6c 111 11 Retinad

KT1–2p 2 2 2 2 2KT1–2m 2 2 2 6 11KT1–3m 4 2 6c 11 11KT1–4m 1 2 6c 111 11 Spleen,d sm intestd

KT1–5m 3 2 6c 111 11 Retinad

K6 expression in mouse skin 2 111 11 11 Does not apply

K6 expression in human skin 2 111 11 11 Does not applya Expression was assessed using adult mouse tissues incubated with X-gal staining solution, paraffin-embedded, and sectioned for light

microscopy. Key: 2, no expression; 6, very sproradic expression; 1, modest but consistent expression; 11, moderately strong expression; 111,very strong expression.

b Wounded tissues (skin and oral mucosa) were examined at 24 h following full-thickness injury (see “Experimental Procedures”).c Expression is very sporadic and restricted to a very small number of outer root sheath keratinocytes in occasional hair follicles (fewer than

1:100).d Expression in these other tissues is sporadic as well, with only a small subset of cells positive for b-galactosidase activity. Abbreviations: esoph,

esophagus; kid, kidney; trach, trachea; pericard, pericardium; sm intest, small intestine.

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ting (data not shown), as well as by enzymatic assays per-formed in soluble extracts prepared from intact skin of adulttransgenic mice (Table II). A notable exception occurs in thevibrissae follicles of whisker pads in several KT1 transgeniclines, which show weak LacZ activity in a greater number ofORS keratinocytes (Fig. 1, compare D and D-inset). Expressionremains patchy, however, and is not seen in transgenic linesmade with shorter K6a promoter-based constructs (KT3, KT4;not shown). A survey of other stratified epithelia known to

express K6, such as nail, cornea (limbus), tongue, oral mucosa,esophagus, and forestomach fails to reveal b-galactosidase ac-tivity in the majority of transgenic lines produced (Fig. 1, E–H),albeit with a few exceptions (Table I). Among such exceptionsare lines KT3-2m and KT3-4m, which show b-galactosidaseactivity in a very small subset of epithelial cells in tongue,esophagus, and/or cornea (typically, only 1–3 cells/entire histo-logical section; Table I). The three other lines made with theKT3 construct do not show expression in these epithelia (Table

FIG. 1. Expression of [hK6a 5*]-LacZ transgenes in intact mouse tissues. The sections shown were prepared from adult mouse tissuesincubated with X-gal, embedded, sectioned, and stained with eosin (frames A, B, D–I) or alternatively processed for histochemistry with anti-mouseK6 followed by a peroxidase conjugate (frames C, D9–I9). A, KT2-2m trunk skin; B, KT1-3m trunk skin; the arrow points to a single b-galactosidasepositive keratinocyte in the outer root sheath of a hair follicle (F); C, KT2-2m tail skin, K6 immunostaining; the outer root sheath of hair follicles(F) is strongly positive, while the epidermis (EPI) shows only background staining (* marks sebaceous glands); D, KT1-3m whisker pad skin; thearrow depicts many b-galactosidase-positive keratinocytes in a vibrissae follicle (V); D (inset), KT1-1p whisker pad skin, showing a b-galactosidase-negative vibrissae follicle (V); E and E9, KT2-2m nail tissue; the K6-positive epidermis in the nail fold (arrowheads) shows no b-galactosidaseactivity; M, nail matrix; F and F9, KT3-3m eye tissue; the K6-positive conjunctival (C) epithelium and limbus area of the cornea (L) are bothnegative for b-galactosidase; G and G9, KT1-4m tongue and H and H9 KT1–1p esophagus, in both these cases the suprabasal layers of the epitheliaare strongly positive for K6 and yet shown no b-galactosidase activity; P, filiform papillae; I and I9, KT1-3p retinal epithelium; the arrow depictsa transgene-positive ganglion cell in this K6-negative tissue. In A, B, C, E, F, G, and H the arrowheads highlight the interface between the stratifiedepithelium and underlying connective tissue. Bars 5 1 mm.

TABLE IIQuantitation of b-galactosidase activity in transgenic mouse skin extracts

Promoter TG linea Intact trunk skinb Wounded skinc TPA-treated skin RA-treated skin

milliunits/mm2 skin1.0 kb KT3–2p 0.22 0.60 NDd ND

KT3–1m 0.03 0.24, 0.41 ND NDKT3–2m 0.13 0.55 ND NDKT3–3m 0.13 0.67 1.48 1.81KT3–4m 0.04 0.52, 0.74 0.70 1.17

2.5 kb KT2–1p 0.11 0.43, 0.58 0.46 0.89KT2–2m 0.08 1.14, 0.68, 1.69 0.69 0.91

5.2 kb KT1–1p 0.09, 0.10 3.30 1.77 3.74KT1–3m 0.08 2.50, 2.64, 2.50 2.94 3.68KT1–4m 0.02 1.16, 1.85 ND NDKT1–5m 0.06 1.91, 1.79 2.62 2.72

Wild-type mice 0.05, 0.06, 0.07e 0.03, 0.14 0.03 0.05a All transgenic animals used were heterozygous at the transgene locus. See Table I for transgene copy number per mouse genome.b Expression was estimated in a 4-mm punch biopsy (full thickness) of skin tissue.c A 2-mm-wide band of skin tissue was dissected at the edge of a full-thickness wound made 48 h earlier.d ND, nondetermined.e Each value represents the average of three different samples obtained from a single mouse. Multiple values correspond to different mice in a

given transgenic line.

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I). Expression of the LacZ transgene is occasionally detected inother types of tissues as well (Table I). Thus, for each K6apromoter construct tested, one or two lines show sporadic ex-pression in the retina, where K6 is not detectable by immuno-histochemistry (Fig. 1, I and I9). Since this “ectopic” expressiondoes not appear to correlate with transgene copy number (Ta-ble I), it appears likely that the transcriptional activity of theLacZ transgenes is somewhat sensitive to their site of integra-tion in the mouse genome.

Induction of Transgene Expression by Injury in StratifiedEpithelia of Transgenic Mice—K6 expression is induced follow-ing injury to the skin and other stratified epithelia (Fig. 2A),and this prompted us to examine transgene expression undersuch conditions. Full-thickness injury to the skin of adult miceinduces lacZ expression in epidermis and hair follicles at thewound edge in most KT1, KT2, and KT3 transgenic lines, but innone of the KT4 lines (Table I). In the responsive lines, b-ga-lactosidase activity occurs in keratinocytes proximal to thewound edge as early as 2.5 h after injury (Fig. 2, B and C) andextends further away from the wound site at later time points,in a pattern analogous to mouse endogenous K6 (Fig. 2D).Immunostaining for the b-galactosidase protein indicates thatit is restricted to suprabasal keratinocytes in wounded skintissue (Fig. 2E). In contrast, mouse endogenous K6 typically

extends down to the basal layer in epidermal tissue at theproximal edge of the wound (Fig. 2A), underscoring a potentialdifference in the regulation of mouse endogenous K6 and ourhuman K6a promoter-based transgenes (see Ref. 8 for similarobservations when using the bovine K6b promoter in trans-genic mice). Induction of LacZ expression also occurs afterinjury to other stratified epithelia, including oral mucosa (Fig.2F; Table I), cornea (Fig. 2G), tongue (not shown), and foot padepidermis (Fig. 2H), with all but the shortest transgene (KT4).Thus, these observations establish that the critical informationrequired to mediate rapid induction following injury is con-tained within the proximal 59-upstream sequence of the humanK6a gene. They further suggest that the signaling pathwaysinvolved in K6 activation after injury are likely to be related, ifnot the same, in these four different stratified epithelia.

The histochemistry findings are supported by b-galactosid-ase enzymatic assays performed on soluble extracts preparedfrom wounded skin tissue, which also reveals differences in theextent of transgene induction depending upon the amount ofK6a 59-upstream sequence involved. At 48 h after skin injury,KT1 transgenic mice show a greater induction of b-galactosid-ase activity compared with KT2 and KT3 mice (Table II). Weselected transgenic lines showing strong expression (KT1-3m,KT2-2m, KT3-3m) to compare the extent of b-galactosidase

FIG. 2. Inducible expression of LacZ transgene in adult transgenic mouse epithelia. The sections shown were prepared from adultmouse tissues incubated with X-gal, embedded, sectioned, and stained with eosin (frames C, D, F, G, I–M, L9) or alternatively processed forhistochemistry with anti-mouse K6 (frames A, I9, J9, K9, M9) or anti-b-galactosidase (E) followed by a peroxidase conjugate. A, control adult mouseskin at two days after injury, showing a strong induction of K6 at the wound edge and in migrating epidermis; F, hair follicle; B, Macroscopic viewof b-galactosidase positive tissue at the edge of a 3-h wound site (see arrows) in a KT2-2m mouse; C, cross-section of the paraffin-embedded skintissue shown in frame B; several b-galactosidase-positive keratinocytes are seen at the edge of the wound site (depicted with an arrow); D, KT3-4mtrunk skin at 2 days after injury; note the strong b-galactosidase activity in epidermis at and away from the wound edge (arrow depicts the woundsite; F, hair follicle); E, KT3-4m trunk skin at 1 day after injury, showing immunostaining for the b-galactosidase protein in suprabasal epidermisat the wound edge (arrow depicts the wound site, and dots depict the position of the dermo-epidermal interface); F, oral mucosa at 1 day after injuryin a KT3-4m mouse (arrow depicts wound site); G, corneal epithelium at 1 day after injury in a KT1-1p mouse (arrow depicts wound site); H,macroscopic view of b-galactosidase-positive tissue at the edge of a 4-h wound site (arrowheads) and a 24-h wound site (short arrows) in the footpad skin of a KT2-2m mouse; I and I9, PMA-treated skin in a KT1-1p mouse, showing positive staining for b-galactosidase and K6; J and J9,RA-treated skin in a KT3-3m mouse, showing positive staining for b-galactosidase and K6; K and K9, DNFB-treated skin in a KT2-2m mouse,showing staining for b-galactosidase and K6; L and L9, weak and sporadic expression of b-galactosidase in chemically induced skin papillomas inKT1-3m and KT1-4m mice; M and M9, trunk skin in a double KT2-2m/hK16 transgenic mouse, show strong K6 expression but virtually nob-galactosidase expression. Unless indicated otherwise, the arrowheads highlight the interface between the epithelium and underlying connectivetissue. Bars 5 1 mm.

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induction as a function of time after skin injury. The dataobtained (Fig. 3) confirmed the rapid induction of the threetypes of transgene in wound edge tissue. However, peak enzy-matic activities were reached at a later time and were three tofive times as large in KT1 mice compared with KT2 and KT3mice (Fig. 3). These data suggest the presence of enhancerelements sensitive to injury and located upstream from 22500bp in the 59-upstream sequence of K6a.

Induction of Transgene Expression by Other Acute Stimuli inthe Skin of Adult Transgenic Mice—Topical application of thephorbol ester PMA and of all-trans-retinoic acid (RA), bothknown to induce K6 expression in mouse epidermis (e.g. Refs. 8and 13), also result in LacZ induction in [hK6a 59]-LacZ trans-genic mice (Fig. 2, I and I9, J, and J9). As following injury, theextent of b-galactosidase enzymatic activity in extracts pre-pared from skin treated with PMA or RA is clearly greater inKT1 transgenic lines than in KT2 and KT3 lines (Table II).Under the treatment regimens tested, RA appears strongerthan PMA in its ability to induce LacZ expression (note thatunlike PMA, treatment with RA significantly alters terminaldifferentiation of skin keratinocytes; see Ref. 13). We alsotested whether DNFB, a potent contact allergen that triggers adelayed-type hypersensitivity reaction (20), could induce trans-gene expression. We found that challenging presensitized skinwith a second application of DNFB to a distinct body site causesa modest LacZ induction in many KT1 transgenic lines (Fig. 2,K and K9). Collectively, our data demonstrate that the 59-upstream region of the human K6a gene contains sufficientregulatory information for its chemical induction in adulttransgenic mouse skin using agents that produce enhancedproliferation (via PMA), altered differentiation (via RA), or acontact dermatitis-like reaction (via DNFB).

We next examined the activity of the [hK6a 59]-LacZ trans-genes in contexts featuring chronic hyperproliferation and al-tered differentiation in adult mouse skin. First, we applied thetwo-step 7,12-dimethylbenz[a]anthracene-12-O-tetradecanoyl-phorbol-13-acetate skin carcinogenesis protocol (21) to produceskin papillomas in the various lines of transgenic mice. Asexpected (22), abundant expression of K6 occurs in premalig-nant papilloma lesions produced in our various lines of trans-genic mice (data not shown). Somewhat surprisingly, a rela-

tively small number of keratinocytes express the transgene infully developed papillomas isolated from KT1 and KT2 trans-genic mice, and the LacZ-positive keratinocytes tend to belocated in the uppermost portion of the much thickened epider-mis (Fig. 2, L and L9). Second, we took advantage of K16-overexpressing transgenic mice available in our laboratory toproduce double-transgenic animals via matings with KT2-2mtransgenic mice (Table I). A particular line of transgenic micecontaining 8–10 copies of the full-length human K16 gene(5-7-K16) develops striking lesions in hair follicle ORS andepidermis in the first week after birth, coinciding with theemergence of fur (17). As expected, double-transgenic micedeveloped similar skin lesions affecting the hair follicle ORSand adjacent epidermis in the first week after birth. However,only patchy LacZ transgene expression could be evidenced inthe skin of various body sites in these mice, even though mouseendogenous K6 was present at high levels (Fig. 2, M and M9).We verified that the transgene retained its ability to respond toacute skin injury in the hK16-LacZ double transgenic mice(data not shown). Together with the data gathered on chemi-cally induced skin papillomas, these findings suggest up to 5.2kb of proximal 59-upstream sequence from the human K6a genemay not contain sufficient information for its sustained expres-sion in contexts akin to chronic hyperproliferative diseases.

DISCUSSION

The Organization of Regulatory Elements in the K6a GeneAppears Unique among Skin Keratin Genes—Several of thekeratin genes are expressed in a stable and predictable fashionin well defined epithelial contexts (5, 7). In normal interfollicu-lar epidermis and a few other cornifying epithelia, for instance,the K5-K14 and K1-K10 genes are expressed in a pairwise andconstitutive fashion in the progenitor and differentiating lay-ers, respectively (23–26). In striking contrast, the K6 isoformgenes show a complex regulation with constitutive and induc-ible components in various stratified epithelia, such that thereis no obvious relationship between K6 expression and a definedprogram of terminal differentiation (see Refs. 7 and 14). Yet,the predicted genomic structure and amino acid sequence of thehuman K6 isoform genes are very related to K5 (a type IIkeratin as well), and accordingly these have been postulated tooriginate from a common ancestral gene (4, 27). Since therelevant gene duplication event, however, the regulation ofthese genes has diverged significantly more than their codingsequences (28). Byrne and Fuchs (18) showed that 6 kb of59-upstream region from the human K5 gene can direct theexpression of a LacZ reporter in a tissue-specific fashion intransgenic mice. Similar results were obtained with the humantype I K14 and K10 genes (29, 30), but not with the type II K1gene (31), whose faithful regulation seems to necessitate se-quences located outside of the proximal 59-upstream sequence(32). Here, we show that the proximal 5.2 kb of 59-upstreamsequence from the dominant K6 isoform gene in human skin,K6a (4), does not support consistent expression of a heterolo-gous reporter sequence at a detectable level in any tissue ofadult transgenic mice (with the potential exception of vibrissae;see below). In separate studies, we found that the presence ofthe 39-untranslated region of the human K6a gene in the con-text of the KT1 and KT2 transgene constructs did not alter theexpression pattern of a distinct coding sequence (a mutant K6acDNA) in transgenic mice (33). We therefore conclude thatwhen assessed in transgenic mice, the constitutive aspect ofhuman K6a expression necessitates sequences that are located:(i) upstream from the proximal 5.2 kb of 59-upstream sequence;(ii) distal to the 39-noncoding region; and/or (iii) in introns, as isthe case for the simple epithelial K18 gene (34). The organiza-tion of regulatory sequence elements in the human K6a gene

FIG. 3. Kinetics of b-galactosidase induction after injury invarious transgenic mouse lines. Two mice were used for each trans-genic line tested. Skin tissue at the wound edge was sampled at 0, 4 h,1 day, and 3 days after injury in one mouse and at 0, 8 h, 2 days, and 5days in the other mouse. b-Galactosidase activity was measured inextracts prepared from a 4-mm punch biopsy of wounded skin. Opentriangles, KT1-3m line; closed circles, KT2-2m line; open circles,KT3-3m line.

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thus appears distinct from that documented for the evolution-ary related K5 gene as well as other major keratin genes thatare constitutively expressed in skin epithelia.

More consistent expression of the KT1 transgene occurs invibrissae follicles, although it still represents a small fraction ofthe K6-positive tissue. This may imply that the regulatorysequences involved in directing constitutive expression of K6are somewhat distinct between hair follicles and vibrissae.Alternatively, however, it could be that the KT1 transgeneactivity is “constantly induced” at a low level by the mildfrictional trauma incurred due to the frequent rubbing of thisarea associated with grooming. Of all the transgene constructstested in our studies, indeed, the KT1 was the most responsiveto trauma.

The results reported here contrast with those reported forthe bovine K6b gene, which encodes a keratin protein mostrelated to human K6 in its predicted amino acid sequence andexpression pattern (the BK6b gene was originally designatedBKIV*; see Ref. 8). Two groups observed a near-tissue-specificexpression of heterologous coding sequences in transgenic micewhen using either 5.2 or 8.8 kb of 59-upstream sequence fromthe BK6b gene (8, 35). The occurrence of such significant dif-ferences is surprising, given the extensive homology in theproximal 59-upstream sequences of the human K6a, K6b, andbovine K6b genes (data not shown; Ref. 38). Multiple K6 iso-form genes have been identified in both the human and bovinegenomes (4, 36, 37), and we do not know whether the bovineK6b gene is the actual ortholog of human K6a. This notioncould explain the differences observed in the activity of the59-upstream sequence of these genes in transgenic mice. Anin-depth comparison of the promoter sequences of these genesshould provide significant insights into the unique aspects ofthe regulation of the human K6a gene.

The keratin 6 gene(s) are co-expressed with the K16 and/orK17 genes as type I keratin partners in stratified epitheliaunder basal or challenged conditions (see Introduction). Wepreviously reported that a full-length genomic clone (11 kb)containing the entire human K16 gene yielded cell type-specificexpression in the trunk skin of transgenic mice under bothbasal and injury conditions (17), but not in specialized skinepithelia such as foot pad epidermis and nail matrix. As thesestudies did not address the contribution of the various seg-ments of the human K16 gene to the pattern of expressionobserved in transgenic mice, the organization of regulatorysequences in the human K6a and K16 genes can not be com-pared at the present time.

Role of the Proximal 59-Upstream Sequence in K6a GeneExpression after Various Acute Stimuli—We demonstratedhere that the proximal 960 bp of 59-upstream sequence in thehuman K6a gene successfully mediates the rapid induction of aheterologous reporter gene in adult transgenic mouse skinafter acute injury or treatment with appropriate chemical in-ducers, while the proximal 550-bp segment can not. At leastwhen studied in transgenic mice, therefore, we conclude thatcis-acting sequences located between 2550 and 2960 bp in thehuman K6a gene are necessary for its induction when subject-ing stratified epithelia to a variety of acute stimuli (injury,12-O-tetradecanoylphorbol-13-acetate, RA, DNFB). Moreover,these regulatory sequences are at least partly distinct fromthose underlying its constitutive expression in the relevantepithelia. Whether these inducible elements activate transcrip-tion by acting directly on core promoter elements or alterna-tively by negating a repressor element located within the prox-imal 550-bp segment remains to be defined. Moreover, givenour observation that the product of the KT3 transgene is spa-tially restricted to the suprabasal layers after its induction

(Fig. 2), as is the case for the K6 isoforms after injury to humanskin (11), we also conclude that the critical elements controllingthe cell type specificity of human K6a expression are likely tobe present within the proximal 960 bp of its 59-upstream se-quence. These data extend the findings of Ramirez et al. (8),who observed an induction of a LacZ reporter transgene fea-turing 8.8 kb of 59-upstream sequence from the bovine K6b

gene after treatment with 12-O-tetradecanoylphorbol-13-ace-tate and RA and after injury to the skin. On the other hand, ourconclusions differ from those reached by Jiang et al. (39, 40),who found that the proximal 390 bp of 59-upstream sequencefrom the human K6b gene conferred positive and cell type-specific expression of a CAT reporter in human keratinocytes inculture, a context that allegedly mimicks hyperproliferation(14). The “promoter region” of many keratin genes has beenfound to behave differently when transfected in cultured celllines compared with when stably integrated within the mousegenome (e.g. Refs. 18, 34, and 41), a notion that may be at playhere. Other explanations for this discrepancy include the ex-istence of distinct regulatory mechanisms for human K6a andK6b (see Ref. 4) or alternatively, the potential presence ofstrong silencer element(s) located between 2390 bp and 2550bp in both these genes.

We observed a much stronger induction of transgene expres-sion following acute chemical induction or injury to adult trans-genic mice bearing a construct featuring 5.2 kb of human K6a59-upstream sequence compared with those having shorter 59sequences (Table II; Fig. 3). For each acute challenge tested(PMA, RA, injury), indeed, the extent of LacZ induction inmouse skin showed a similar dependence upon the amount of59-upstream sequence in the transgene. This notion suggeststhat the relevant regulatory elements in the proximal corepromoter are subject to positive regulation by powerful en-hancer element(s) located between 22500 and 25200 bp in thehuman K6a gene. Our data also suggest that the molecularmechanisms that trigger K6 induction after acute stimuli inskin may differ to some extent from those underlying its sus-tained expression in chronic lesions such as those typical ofpsoriasis and benign and malignant neoplasia. Further char-acterization of the human K6a gene in transgenic mice shouldenable us to define the identity and mode of action of thevarious functional elements involved in controlling the complexregulation of this gene.

It should been emphasized that the results reported hereapply to post-natal mouse skin and that our interpretation ofthe expression pattern is based on the comparison of the dis-tribution of the transgene product with that of mouse K6 pro-tein(s). Transient expression of K6 has been detected in epider-mis at a late stage of human fetal development (week 36; seeRef. 7). Studies are in progress to examine whether the [59hK6a]-LacZ transgene is expressed pre-natally in developinghair follicles or epidermis in our lines of transgenic mice. Atanother level, a close examination of the pattern of [59 hK6a]-LacZ transgene expression in adult transgenic mouse skin sug-gests that after induction not all suprabasal keratinocytesshow a b-galactosidase-positive nucleus, whereas the majorityof them stain positive for mouse K6 protein(s) under the sameconditions (e.g. Fig. 2). As apparent from previous transgenicmouse studies (18), the b-galactosidase protein may be rela-tively short-lived in skin keratinocytes, even when targeted tothe nucleus (this study). Given that keratin proteins are verystable in epithelial cells (7), a survey of the mouse K6 mRNA(s)distribution would provide a more suitable reference againstwhich to compare the distribution of the transgene product. Ina parallel set of transgenic mouse studies involving the samepromoter sequences, we found that after induction by PMA

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application (33) or injury (data not shown), a Myc epitope-tagged transgenic keratin protein shows more consistent ex-pression in suprabasal epidermis. A characterization of themouse K6 isoform family has yet to be completed and shouldenable the design of suitable probes for the specific detection ofK6 mRNA(s). These issues are of significant importance for ourunderstanding of the control of de novo keratin gene transcrip-tion at a spatial and temporal levels in stratified epitheliasubjected to various types of challenges. This information isalso needed to better exploit the 59-upstream region of thehuman K6a gene for inducible expression or inducible generearrangements in stratified epithelia of transgenic mice.

Is K6 Induction a Faithful Marker of Hyperproliferation inStratified Epithelia?—Induction or enhancement of K6 andK16 expression often accompanies enhanced mitotic activity instratified epithelia (9), such that the former has often beentaken as direct evidence for the latter. Various lines of evidencesuggest, however, that induction of K6/K16 expression andenhanced cell proliferation in stratified epithelia may be trig-gered by distinct signaling pathways. Thus, the expression ofK6 and K16 is restricted to post-mitotic, suprabasal keratino-cytes under conditions featuring enhanced proliferation in skin(e.g. psoriasis, carcinoma, and after injury; see Refs. 10, 11, and42). In addition, it has been shown that the population ofsuprabasal keratinocytes expressing K6 at the wound edgefollowing skin injury clearly extends away from a narrowerzone of tissue containing basal keratinocytes with enhancedmitotic activity (see Ref. 2). The evidence introduced hereshows that consistent with the rapid appearance of K6 of theK16 proteins in wound edge epidermis after injury to human(11) and mouse skin (data not shown), the induction of the[hK6a 59]-LacZ transgenes occurs within 2.5 h after injury totransgenic mouse epidermis. Yet, an enhancement of mitoticactivity in the basal epidermal layer at the wound edge is firstdetectable at ;20–30 h after injury to mouse and human skin(see Refs. 2 and 11 and references therein). Taken together,these observations point to the existence of significant differ-ences at a spatial and temporal levels with regards to K6induction and enhanced keratinocyte proliferation at thewound edge. This evidence derived from in vivo studies corrob-orate previous findings dissociating K6 expression from mitoticactivity in ex vivo cultures of epidermal keratinocytes andcorneal epithelial cells (14, 42, 43). At another level, we alsofound that expression of the KT2 transgene was sporadic atbest in chemically induced skin papillomas as well as in thechronic skin lesions of K16-overexpressing transgenic mice. Yetin both circumstances the skin lesions feature abundant K6protein levels and a lymphocytic infiltration in the context of amarkedly thickened, hyperproliferative epidermis. Based onthe frequent presence of b-galactosidase activity in the upper-most layers of lesional epidermis (e.g. Fig. 2M), it appearslikely that these lesions featured transgene expression at anearlier stage of their development. However, the constructtested (KT2) apparently lacks the regulatory sequences re-quired for a sustained expression in a K6-like fashion inchronic hyperproliferative lesions. Collectively, these observa-tions provide strong evidence that enhanced K6 expression canbe dissociated from enhanced keratinocyte proliferation instratified epithelia in vivo, suggesting that the regulatory path-ways involved are at least partially distinct.

Acknowledgments—We are grateful to S. Brust and A. Chen (JohnsHopkins University Transgenic Core Facility) for the production of

transgenic mice and Dr. K. McGowan for his advice and assistance. Wealso thank Dr. D. Paulin for providing the nls-LacZ reporter sequence,Dr. D. Roop for providing an antiserum to mouse K6, and Drs. E.Colucci-Guyon and C. Byrne for their advice.

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Kenzo Takahashi and Pierre A. Coulombein Stratified Epithelia of Transgenic Mice

Defining a Region of the Human Keratin 6a Gene That Confers Inducible Expression

doi: 10.1074/jbc.272.18.119791997, 272:11979-11985.J. Biol. Chem. 

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