(Received for publication, October 15, 1996, and in revised form, January 28, 1997)
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
epithelia triggers a repair response involving the rapid induction of
several genes, including keratin 6 (K6). The signaling pathways and
mechanisms presiding over this induction in keratinocytes at the wound
edge remain to be defined. We reported previously that of the multiple genes encoding K6 isoforms in human, K6a is dominant in skin epithelia (Takahashi, K., Paladini, R., Coulombe, P. A. (1995) J. Biol. Chem. 270, 18581-18592). Using bacterial LacZ as a
reporter gene in transgenic mice, we show that the proximal 5.2 kilobases of 5-upstream sequence from the K6a gene fails to direct
sustained expression in any adult tissue, including those where K6 is
constitutively expressed (e.g. hair follicle, nail, oral
mucosa, tongue, esophagus, forestomach). In contrast, the proximal 960 base pairs of 5
-upstream sequence suffice to mediate an induction of
-galactosidase expression in a near-correct spatial and temporal
fashion after injury to epidermis and other stratified epithelia.
Transgene expression also occurs following topical application of
phorbol esters, all-trans-retinoic acid, or
2-4-dinitro-1-fluorobenzene, all known to induce K6 expression in
skin. Our data show that critical regulatory sequences for this
inducibility are located between
960 and
550 bp in the 5
-upstream
sequence of K6a and that their activity is influenced by enhancer
element(s) located between
2500 and
5200 base pairs. These findings
have important implications for the control of gene expression after
injury to stratified epithelia.
Injury to skin triggers a repair response aimed at restoring epithelial continuity and barrier function. The activity of several genes encoding intracellular, cell surface, and secreted proteins is rapidly modulated in the epithelial and mesenchymal cells involved in this response (1). Keratins 6, 16, and 17, the gap junction protein connexin 26, the receptor for the urokinase-type plasminogen activator, and various proteases are induced in wound edge keratinocytes within hours after injury, and their subsequent accumulation correlates with major changes in keratinocyte cytoarchitecture that precede the onset of migration toward the wound site (2). While the significance of these changes remains to be elucidated, the study of the regulation of the corresponding genes offers an opportunity to decipher the molecular mechanisms underlying the onset of the wound repair response in stratified epithelia. Indeed, even though the levels of several potent growth factors are greatly elevated in the wound site early after injury to the skin (reviewed in Ref. 3), those playing a critical role in this vital homeostatic response remain to be identified.
We recently cloned several human genes and cDNAs predicted to
encode highly related keratin 6 (K6) isoforms (4). K6 (56 kDa) is a
type II keratin that belongs to the superfamily of intermediate filament proteins and is ususally co-expressed with one or two type I
keratins, K16 and K17 (5, 6). The K6 isoforms show a complex pattern of
expression in epithelia, with constitutive and inducible components
(7). They are normally found in the outer root sheath
(ORS)1 of hair follicles, in glandular
tissues, in tongue, gingiva and oral mucosa, esophagus, forestomach,
and certain reproductive tract epithelia (e.g. Ref. 8). With
the exception of palm and sole, K6 is not expressed in normal
interfollicular epidermis (5, 7, 8). The K6 isoforms are better known
for their much enhanced expression during hyperproliferation and
abnormal differentiation in stratified epithelia (4, 9, 10). Thus, K6
and K16 are induced in wound edge keratinocytes as early as 4-6 h
after injury to human skin and disappear after closure (11, 12). K6
expression is induced as well in a variety of diseases affecting
complex epithelia, such as infections, squamous metaplasia, carcinoma,
and chronic hyperproliferative disorders, including psoriasis (9, 10).
In these conditions, K6 expression may be very abundant, but is usually
restricted to the suprabasal compartment of the epithelium (10). In
mouse skin, K6 expression is induced after topical application of a
variety of chemicals (e.g. phorbol esters, retinoic acid;
see Ref. 13). K6 induction also occurs in primary cultures of
mitotically active keratinocytes from epidermis, esophagus, trachea,
and cornea (7, 14). Understanding the regulation of K6 gene expression
is thus of great interest at various levels, one being the control of
gene expression in contexts such as wound repair, psoriasis, and
carcinoma. Using a transgenic mouse approach, we report here on the
identification of a segment of 5-upstream sequence in the human K6a
gene that is both necessary and sufficient for the inducible expression of an heterologous reporter gene in adult mouse epithelia.
Our
starting template was the human K6a gene, the dominant K6 isoform in
hair follicle outer root sheath, foot sole epidermis, and skin squamous
carcinoma 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 5-upstream sequence from the
translation initiation codon were isolated from the human K6a gene
(GenBank accession numbers L42575-L42583[GenBank][GenBank][GenBank][GenBank][GenBank][GenBank][GenBank][GenBank][GenBank]; see Ref. 4) by restriction
digestion and subcloned into a LacZ expression cassette. We used a LacZ
coding sequence (plasmid pCH110) modified to contain a nuclear
localization sequence at its 5
-coding end in addition to the SV40
poly(A) sequence at its 3
end (15). The four transgene constructs
devised are as follows: KT1, [5.20-kb hK6a 5
-upstream
sequence]-LacZ; KT2: [2.55-kb hK6a 5
-upstream sequence]-LacZ; KT3,
[0.96-kb hK6a 5
-upstream sequence]-LacZ; KT4, [0.55-kb hK6a
5
-upstream sequence]-LacZ.
Transgenic mice were produced by pronuclear injection of DNA constructs in single cell C57B6/BalbC3 embryos (16). Founders were identified by Southern blotting of genomic DNAs using a probe to the coding portion of LacZ. Transgenic lines were established by matings in the C57B6/BalbC3 mixed background (Jackson Laboratories). Double-transgenic mice were produced by mating the previously described PC5-7-K16 transgenic mice, which contain 8-10 copies of the full-length human K16 gene (17), with KT2-2m transgenic mice (Table I).
|
For -galactosidase histochemistry
in situ, adult mouse tissues were prefixed in 1%
formaldehyde, 0.2% glutaraldehyde (60 min), washed in
phosphate-buffered saline, incubated overnight at 30 °C in a
solution containing 1 mg/ml X-gal, 100 mM sodium phosphate
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-µm sections were counter-stained with eosin. For
immunohistochemistry, Bouin's-fixed tissues were paraffin-embedded,
and 5-µm sections were reacted with antisera directed against mouse
K6 (19) or anti-
-galactosidase (Promega, Madison, WI). Bound primary
antibodies were revealed by a peroxidase-based reaction as recommended
(Kirkegaard and Perry Laboratories, Gaithersburg, MD). For biochemical
analysis, adult mouse skin tissue extracts were prepared by
homogenization in
-galactosidase reporter lysis buffer, and
post-centrifugation supernatants were used for the detection of
-galactosidase enzymatic activity following the manufacturer's
instructions (Promega).
All studies involving animals were reviewed by the Johns
Hopkins University Animal Use and Care Committee. For studies involving skin, adult mice (3-6 months old) were anesthetized with avertin and
their backs epilated 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
other stratified epithelia, adult mice were anesthetized, and short
superficial incisions were made with a sterile scalpel to either foot
pad epidermis, cornea, oral mucosa, or tongue. Tissues were harvested
after 24 h and processed for -galactosidase histochemistry as
described above. For studies involving chemical treatment of skin,
solutions of PMA (phorbol-12-myristate-13-acetate, 150 µl of a 50 µM stock in acetone; Sigma) and
all-trans-retinoic acid (150 µl of a 100 µg/ml stock in
ethanol; Sigma) were applied topically on Nair-epilated skin every 3rd
day for three times. To induce a delayed-type skin hypersensitivity
reaction, mice were sensitized with an application of 25 µl of 0.25%
2-4-dinitro-1-fluorobenzene (DNFB) at the base of the tail and
challenged 5 days later by application of 10 µl of the same solution
onto the dorsal neck area as described (20). The mice were sacrificed
and the skin processed for
-galactosidase histochemistry on the next
day. Skin papillomas were induced using the two-step chemical
carcinogenesis procedure (21), involving initiation with
7,12-dimethylbenz[
]anthracene and promotion with PMA for 11 weeks.
Papillomas were harvested 1 month after cessation of treatment and
processed for analysis. In all experiments involving chemical inducers,
controls consisted in application of the vehicle only.
Table I lists
the transgenic lines produced for each of the constructs and reports on
transgene expression assessed by -galactosidase histochemistry
in situ. None of the constructs, including KT1 (5.2 kb of
K6a 5
-upstream sequence), shows consistent expression in the ear,
trunk, tail, or paw skin of adult transgenic mice (Fig.
1A). In KT1, KT2, and KT3 lines, occasional
ORS keratinocytes display
-galactosidase activity in a subset of
hair follicles (Table I; Fig. 1A). Generally, fewer that
three to five follicles show sporadic X-gal staining in the ORS in a
typical section (1-2 cm wide) of adult skin tissue (Fig.
1B). In contrast, endogenous K6 is easily detected in mouse
hair follicles (Fig. 1C). These findings are supported by
immunostainings using antibodies against the
-galactosidase protein,
by Northern blotting (data not shown), as well as by enzymatic assays
performed in soluble extracts prepared from intact skin of adult
transgenic mice (Table II). A notable exception occurs
in the vibrissae follicles of whisker pads in several KT1 transgenic
lines, which show weak LacZ activity in a greater number of ORS
keratinocytes (Fig. 1, compare D and D-inset). Expression remains patchy, however, and is not seen in transgenic lines
made 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
-galactosidase activity in the majority of
transgenic lines produced (Fig. 1, E-H), albeit with a few
exceptions (Table I). Among such exceptions are lines KT3-2m and
KT3-4m, which show
-galactosidase activity in a very small subset of
epithelial cells in tongue, esophagus, and/or cornea (typically, only
1-3 cells/entire histological section; Table I). The three other lines
made with the KT3 construct do not show expression in these epithelia
(Table I). Expression of the LacZ transgene is occasionally detected in
other types of tissues as well (Table I). Thus, for each K6a promoter
construct tested, one or two lines show sporadic expression in the
retina, where K6 is not detectable by immunohistochemistry (Fig. 1,
I and I
). Since this "ectopic" expression
does not appear to correlate with transgene copy number (Table I), it
appears likely that the transcriptional activity of the LacZ transgenes
is somewhat sensitive to their site of integration in the mouse
genome.
|
K6 expression is induced following injury to
the skin and other stratified epithelia (Fig.
2A), and this prompted us to examine transgene expression under such conditions. Full-thickness injury to
the skin of adult mice induces lacZ expression in epidermis and hair
follicles at the wound edge in most KT1, KT2, and KT3 transgenic lines,
but in none of the KT4 lines (Table I). In the responsive lines,
-galactosidase activity occurs in keratinocytes proximal to the
wound edge as early as 2.5 h after injury (Fig. 2, B
and C) and extends further away from the wound site at later time points, in a pattern analogous to mouse endogenous K6 (Fig. 2D). Immunostaining for the
-galactosidase protein
indicates that it is restricted to suprabasal keratinocytes in wounded
skin tissue (Fig. 2E). In contrast, mouse endogenous K6
typically extends down to the basal layer in epidermal tissue at the
proximal edge of the wound (Fig. 2A), underscoring a
potential difference in the regulation of mouse endogenous K6 and our
human K6a promoter-based transgenes (see Ref. 8 for similar
observations when using the bovine K6
promoter in transgenic mice).
Induction of LacZ expression also occurs after injury to other
stratified epithelia, including oral mucosa (Fig. 2F; Table
I), cornea (Fig. 2G), tongue (not shown), and foot pad
epidermis (Fig. 2H), with all but the shortest transgene
(KT4). Thus, these observations establish that the critical information
required to mediate rapid induction following injury is contained
within the proximal 5
-upstream sequence of the human K6a gene. They
further suggest that the signaling pathways involved in K6 activation
after injury are likely to be related, if not the same, in these four
different stratified epithelia.
The histochemistry findings are supported by -galactosidase
enzymatic assays performed on soluble extracts prepared from wounded
skin tissue, which also reveals differences in the extent of transgene
induction depending upon the amount of K6a 5
-upstream sequence
involved. At 48 h after skin injury, KT1 transgenic mice show a
greater induction of
-galactosidase activity compared with KT2 and
KT3 mice (Table II). We selected transgenic lines showing strong
expression (KT1-3m, KT2-2m, KT3-3m) to compare the extent of
-galactosidase induction as a function of time after skin injury.
The data obtained (Fig. 3) confirmed the rapid induction
of the three types of transgene in wound edge tissue. However, peak
enzymatic activities were reached at a later time and were three to
five times as large in KT1 mice compared with KT2 and KT3 mice (Fig.
3). These data suggest the presence of enhancer elements sensitive to
injury and located upstream from
2500 bp in the 5
-upstream sequence
of K6a.
Induction of Transgene Expression by Other Acute Stimuli in the Skin of Adult Transgenic Mice
Topical application of the phorbol
ester PMA and of all-trans-retinoic acid (RA), both known to
induce K6 expression in mouse epidermis (e.g. Refs. 8 and
13), also result in LacZ induction in [hK6a 5]-LacZ transgenic mice
(Fig. 2, I and I
, J, and J
). As
following injury, the extent of
-galactosidase enzymatic activity in
extracts prepared from skin treated with PMA or RA is clearly greater
in KT1 transgenic lines than in KT2 and KT3 lines (Table II). Under the
treatment regimens tested, RA appears stronger than PMA in its ability
to induce LacZ expression (note that unlike PMA, treatment with RA
significantly alters terminal differentiation of skin keratinocytes;
see Ref. 13). We also tested whether DNFB, a potent contact allergen
that triggers a delayed-type hypersensitivity reaction (20), could
induce transgene expression. We found that challenging presensitized
skin with a second application of DNFB to a distinct body site causes a
modest LacZ induction in many KT1 transgenic lines (Fig. 2,
K and K
). Collectively, our data demonstrate
that the 5
-upstream region of the human K6a gene contains sufficient
regulatory information for its chemical induction in adult transgenic
mouse skin using agents that produce enhanced proliferation (via PMA),
altered differentiation (via RA), or a contact dermatitis-like reaction
(via DNFB).
We next examined the activity of the [hK6a 5]-LacZ transgenes in
contexts featuring chronic hyperproliferation and altered differentiation in adult mouse skin. First, we applied the two-step 7,12-dimethylbenz[
]anthracene-12-O-tetradecanoylphorbol-13-acetate skin carcinogenesis protocol (21) to produce skin papillomas in the
various lines of transgenic mice. As expected (22), abundant expression
of K6 occurs in premalignant papilloma lesions produced in our various
lines of transgenic mice (data not shown). Somewhat surprisingly, a
relatively small number of keratinocytes express the transgene in fully
developed papillomas isolated from KT1 and KT2 transgenic mice, and the
LacZ-positive keratinocytes tend to be located in the uppermost portion
of the much thickened epidermis (Fig. 2, L and
L
). Second, we took advantage of K16-overexpressing transgenic mice available in our laboratory to produce
double-transgenic animals via matings with KT2-2m transgenic mice
(Table I). A particular line of transgenic mice containing 8-10 copies
of the full-length human K16 gene (5-7-K16) develops striking lesions in hair follicle ORS and epidermis in the first week after birth, coinciding with the emergence of fur (17). As expected,
double-transgenic mice developed similar skin lesions affecting the
hair follicle ORS and adjacent epidermis in the first week after birth.
However, only patchy LacZ transgene expression could be evidenced in
the skin of various body sites in these mice, even though mouse
endogenous K6 was present at high levels (Fig. 2, M and
M
). We verified that the transgene retained its ability to
respond to acute skin injury in the hK16-LacZ double transgenic mice
(data not shown). Together with the data gathered on chemically induced
skin papillomas, these findings suggest up to 5.2 kb of proximal
5
-upstream sequence from the human K6a gene may not contain sufficient
information for its sustained expression in contexts akin to chronic
hyperproliferative diseases.
Several of the keratin genes are
expressed in a stable and predictable fashion in well defined
epithelial contexts (5, 7). In normal interfollicular epidermis and a
few other cornifying epithelia, for instance, the K5-K14 and K1-K10
genes are expressed in a pairwise and constitutive fashion in the
progenitor and differentiating layers, respectively (23-26). In
striking contrast, the K6 isoform genes show a complex regulation with
constitutive and inducible components in various stratified epithelia,
such that there is no obvious relationship between K6 expression and a
defined program of terminal differentiation (see Refs. 7 and 14). Yet,
the predicted genomic structure and amino acid sequence of the human K6
isoform genes are very related to K5 (a type II keratin as well), and
accordingly these have been postulated to originate from a common
ancestral gene (4, 27). Since the relevant gene duplication event,
however, the regulation of these genes has diverged significantly more
than their coding sequences (28). Byrne and Fuchs (18) showed that 6 kb
of 5-upstream region from the human K5 gene can direct the expression
of a LacZ reporter in a tissue-specific fashion in transgenic mice.
Similar results were obtained with the human type I K14 and K10 genes
(29, 30), but not with the type II K1 gene (31), whose faithful
regulation seems to necessitate sequences located outside of the
proximal 5
-upstream sequence (32). Here, we show that the proximal 5.2 kb of 5
-upstream sequence from the dominant K6 isoform gene in human
skin, K6a (4), does not support consistent expression of a heterologous
reporter sequence at a detectable level in any tissue of adult
transgenic mice (with the potential exception of vibrissae; see below).
In separate studies, we found that the presence of the 3
-untranslated
region of the human K6a gene in the context of the KT1 and KT2
transgene constructs did not alter the expression pattern of a distinct
coding sequence (a mutant K6a cDNA) in transgenic mice (33). We
therefore conclude that when assessed in transgenic mice, the
constitutive aspect of human K6a expression necessitates sequences that
are located: (i) upstream from the proximal 5.2 kb of 5
-upstream
sequence; (ii) distal to the 3
-noncoding region; and/or (iii) in
introns, as is the case for the simple epithelial K18 gene (34). The
organization of regulatory sequence elements in the human K6a gene thus
appears distinct from that documented for the evolutionary related K5 gene as well as other major keratin genes that are constitutively expressed in skin epithelia.
More consistent expression of the KT1 transgene occurs in vibrissae follicles, although it still represents a small fraction of the K6-positive tissue. This may imply that the regulatory sequences involved in directing constitutive expression of K6 are somewhat distinct between hair follicles and vibrissae. Alternatively, however, it could be that the KT1 transgene activity is "constantly induced" at a low level by the mild frictional trauma incurred due to the frequent rubbing of this area associated with grooming. Of all the transgene constructs tested in our studies, indeed, the KT1 was the most responsive to trauma.
The results reported here contrast with those reported for the bovine
K6 gene, which encodes a keratin protein most related to human K6 in
its predicted amino acid sequence and expression pattern (the BK6
gene was originally designated BKIV*; see Ref. 8). Two groups observed
a near-tissue-specific expression of heterologous coding sequences in
transgenic mice when using either 5.2 or 8.8 kb of 5
-upstream sequence
from the BK6
gene (8, 35). The occurrence of such significant
differences is surprising, given the extensive homology in the proximal
5
-upstream sequences of the human K6a, K6b, and bovine K6
genes
(data not shown; Ref. 38). Multiple K6 isoform genes have been
identified in both the human and bovine genomes (4, 36, 37), and we do
not know whether the bovine K6
gene is the actual ortholog of human K6a. This notion could explain the differences observed in the activity
of the 5
-upstream sequence of these genes in transgenic mice. An
in-depth comparison of the promoter sequences of these genes should
provide significant insights into the unique aspects of the regulation
of the human K6a gene.
The keratin 6 gene(s) are co-expressed with the K16 and/or K17 genes as type I keratin partners in stratified epithelia under basal or challenged conditions (see Introduction). We previously reported that a full-length genomic clone (11 kb) containing the entire human K16 gene yielded cell type-specific expression in the trunk skin of transgenic mice under both basal and injury conditions (17), but not in specialized skin epithelia such as foot pad epidermis and nail matrix. As these studies did not address the contribution of the various segments of the human K16 gene to the pattern of expression observed in transgenic mice, the organization of regulatory sequences in the human K6a and K16 genes can not be compared at the present time.
Role of the Proximal 5We demonstrated here that the
proximal 960 bp of 5-upstream sequence in the human K6a gene
successfully mediates the rapid induction of a heterologous reporter
gene in adult transgenic mouse skin after acute injury or treatment
with appropriate chemical inducers, while the proximal 550-bp segment
can not. At least when studied in transgenic mice, therefore, we
conclude that cis-acting sequences located between
550 and
960 bp
in the human K6a gene are necessary for its induction when subjecting
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 from those
underlying its constitutive expression in the relevant epithelia.
Whether these inducible elements activate transcription by acting
directly on core promoter elements or alternatively by negating a
repressor element located within the proximal 550-bp segment remains to
be defined. Moreover, given our observation that the product of the KT3
transgene is spatially restricted to the suprabasal layers after its
induction (Fig. 2), as is the case for the K6 isoforms after injury to
human skin (11), we also conclude that the critical elements
controlling the cell type specificity of human K6a expression are
likely to be present within the proximal 960 bp of its 5
-upstream
sequence. These data extend the findings of Ramirez et al.
(8), who observed an induction of a LacZ reporter transgene featuring
8.8 kb of 5
-upstream sequence from the bovine K6
gene after
treatment with 12-O-tetradecanoylphorbol-13-acetate and RA
and after injury to the skin. On the other hand, our conclusions differ
from those reached by Jiang et al. (39, 40), who found that
the proximal 390 bp of 5
-upstream sequence from the human K6b gene
conferred positive and cell type-specific expression of a CAT reporter
in human keratinocytes in culture, a context that allegedly mimicks hyperproliferation (14). The "promoter region" of many keratin genes has been found to behave differently when transfected in cultured
cell lines compared with when stably integrated within the mouse genome
(e.g. Refs. 18, 34, and 41), a notion that may be at play
here. Other explanations for this discrepancy include the existence of
distinct regulatory mechanisms for human K6a and K6b (see Ref. 4) or
alternatively, the potential presence of strong silencer element(s)
located between
390 bp and
550 bp in both these genes.
We observed a much stronger induction of transgene expression following
acute chemical induction or injury to adult transgenic mice bearing a
construct featuring 5.2 kb of human K6a 5-upstream sequence compared
with those having shorter 5
sequences (Table II; Fig. 3). For each
acute challenge tested (PMA, RA, injury), indeed, the extent of LacZ
induction in mouse skin showed a similar dependence upon the amount of
5
-upstream sequence in the transgene. This notion suggests that the
relevant regulatory elements in the proximal core promoter are subject
to positive regulation by powerful enhancer element(s) located between
2500 and
5200 bp in the human K6a gene. Our data also suggest that
the molecular mechanisms that trigger K6 induction after acute stimuli
in skin may differ to some extent from those underlying its sustained expression in chronic lesions such as those typical of psoriasis and
benign and malignant neoplasia. Further characterization of the human
K6a gene in transgenic mice should enable us to define the identity and
mode of action of the various functional elements involved in
controlling the complex regulation of this gene.
It should been emphasized that the results reported here apply to
post-natal mouse skin and that our interpretation of the expression
pattern is based on the comparison of the distribution of the transgene
product with that of mouse K6 protein(s). Transient expression of K6
has been detected in epidermis at a late stage of human fetal
development (week 36; see Ref. 7). Studies are in progress to examine
whether the [5 hK6a]-LacZ transgene is expressed pre-natally in
developing hair follicles or epidermis in our lines of transgenic mice.
At another level, a close examination of the pattern of [5
hK6a]-LacZ transgene expression in adult transgenic mouse skin
suggests that after induction not all suprabasal keratinocytes show a
-galactosidase-positive nucleus, whereas the majority of them stain
positive for mouse K6 protein(s) under the same conditions
(e.g. Fig. 2). As apparent from previous transgenic mouse
studies (18), the
-galactosidase protein may be relatively short-lived in skin keratinocytes, even when targeted to the nucleus (this study). Given that keratin proteins are very stable in epithelial cells (7), a survey of the mouse K6 mRNA(s) distribution would provide
a more suitable reference against which to compare the distribution of
the transgene product. In a parallel set of transgenic mouse studies
involving the same promoter sequences, we found that after induction by
PMA application (33) or injury (data not shown), a Myc epitope-tagged
transgenic keratin protein shows more consistent expression in
suprabasal epidermis. A characterization of the mouse K6 isoform family
has yet to be completed and should enable the design of suitable probes
for the specific detection of K6 mRNA(s). These issues are of
significant importance for our understanding of the control of de novo
keratin gene transcription at a spatial and temporal levels in
stratified epithelia subjected to various types of challenges. This
information is also needed to better exploit the 5
-upstream region of
the human K6a gene for inducible expression or inducible gene
rearrangements in stratified epithelia of transgenic mice.
Induction or enhancement of K6 and K16
expression often accompanies enhanced mitotic activity in stratified
epithelia (9), such that the former has often been taken as direct
evidence for the latter. Various lines of evidence suggest, however,
that induction of K6/K16 expression and enhanced cell proliferation in
stratified epithelia may be triggered by distinct signaling pathways.
Thus, the expression of K6 and K16 is restricted to post-mitotic,
suprabasal keratinocytes under conditions featuring enhanced
proliferation in skin (e.g. psoriasis, carcinoma, and after
injury; see Refs. 10, 11, and 42). In addition, it has been shown that
the population of suprabasal keratinocytes expressing K6 at the wound edge following skin injury clearly extends away from a narrower zone of
tissue containing basal keratinocytes with enhanced mitotic activity
(see Ref. 2). The evidence introduced here shows that consistent with
the rapid appearance of K6 of the K16 proteins in wound edge epidermis
after injury to human (11) and mouse skin (data not shown), the
induction of the [hK6a 5]-LacZ transgenes occurs within 2.5 h
after injury to transgenic mouse epidermis. Yet, an enhancement of
mitotic activity in the basal epidermal layer at the wound edge is
first detectable 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 differences at a
spatial and temporal levels with regards to K6 induction and enhanced
keratinocyte proliferation at the wound edge. This evidence derived
from in vivo studies corroborate previous findings
dissociating K6 expression from mitotic activity in ex vivo
cultures of epidermal keratinocytes and corneal epithelial cells (14,
42, 43). At another level, we also found that expression of the KT2
transgene was sporadic at best in chemically induced skin papillomas as
well as in the chronic skin lesions of K16-overexpressing transgenic
mice. Yet in both circumstances the skin lesions feature abundant K6
protein levels and a lymphocytic infiltration in the context of a
markedly thickened, hyperproliferative epidermis. Based on the frequent presence of
-galactosidase activity in the uppermost layers of lesional epidermis (e.g. Fig. 2M), it appears
likely that these lesions featured transgene expression at an earlier
stage of their development. However, the construct tested (KT2)
apparently lacks the regulatory sequences required for a sustained
expression in a K6-like fashion in chronic hyperproliferative lesions.
Collectively, these observations provide strong evidence that enhanced
K6 expression can be dissociated from enhanced keratinocyte
proliferation in stratified epithelia in vivo, suggesting
that the regulatory pathways involved are at least partially
distinct.
We are grateful to S. Brust and A. Chen (Johns Hopkins University Transgenic Core Facility) for the production of transgenic mice and Dr. K. McGowan for his advice and assistance. We also 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.