(Received for publication, February 2, 1995; and in revised form, July 3, 1995)
From the
The suprabasal keratin 6 (K6) is remarkable among the keratins
as, in addition to being constitutively expressed in different
stratified epithelia, it is induced in epidermis under
hyperproliferative conditions, such as benign or malignant tumors,
psoriasis, and wound healing. In addition, this keratin is also induced
in skin treated with 12-O-tetradecanoylphorbol-13-acetate or
retinoic acid (RA). These characteristics make the study of K6
regulatory elements an especially interesting issue, in particular
because these elements could be useful in designing gene constructs for
the therapy of skin diseases. We have analyzed by mobility shift and
footprinting experiments the cell type-specific enhancer of the bovine
K6 gene (Blessing, M., Jorcano, J. L., and Franke, W. W.(1989) EMBO J. 8, 117-126) and have identified an AP-2-like
element, two AP-1 elements (one of them composite), and a retinoic
acid-responsive element (RARE). Mutagenesis experiments and
cotransfections with retinoic acid receptors show that the RARE
mediates enhancer activation by RA. Chloramphenicol acetyltransferase
assays show that under normal culture conditions, the AP-1 element
retains most of the enhancer transcriptional activity, while the RARE
and AP-2 are weakly active. However, following RA treatment, the AP-1
element is repressed and the RARE is activated, resulting in an overall
stimulation of the enhancer by RA in the BMGE+H cells used in our
study. These results explain in part the complex and sometimes
contradictory response of keratin 6 to hyperproliferative stimuli.
In the last few years, much attention has been drawn toward keratins, since this broad family of cytoskeletal proteins specifically expressed in epithelia has been found to be related to a series of epidermal hereditary diseases (for a recent review on this topic, see (1) ). The keratin family is subdivided into acidic (type I) and basic (type II) proteins. Keratin intermediate filaments are formed by heterodimers containing one molecule of each type (see (2) for a recent review on keratin structure and function). Particular pairs of type I and type II keratins have been correlated with the different routes of epithelial differentiation (3) in such a way that each epithelial cell type expresses a characteristic combination of keratin pairs. This accurately regulated expression pattern suggests that correct keratin expression is necessary to achieve structural integrity and/or correct function of a given epithelium.
Keratin K6 ()is particularly interesting,
because it seems to be regulated in two different ways. (i) It is
constitutively expressed in several internal stratified epithelia, such
as those of the oral cavity (tongue and palate), esophagus, and genital
tract (exocervix and vagina)(4, 5) . It is also
expressed in some areas of the epidermis such as footsoles (4) and in the hair follicle outer root
sheath(6, 7) . (ii) In addition, this keratin is
induced in interfolicular epidermis under hyperproliferative
situations, such as wound healing(8) , psoriasis, tumors, and, in vitro, in cultured epidermal cells (see, e.g., (9) and (10) ). It is also induced in vivo by
agents provoking epidermal hyperplasia, such as TPA and
RA(11, 12, 13, 14) . Thus, the
expression of this keratin seems to be regulated both in a constitutive
and in an inducible manner. It is precisely this capability to be
induced by topical treatments that makes the K6 gene attractive for the
development of gene therapy protocols for skin diseases. Elucidation of
the mechanisms that mediate K6 induction would allow activation and
deactivation at will of the expression of genes under the control of
the K6 regulatory region.
We have shown previously that the enhancer
of the bovine keratin K6 gene (BK6
, formerly referred to as
BK IV*), located between -247 and -647 from the translation
start, is able to drive the cell type-specific expression of a
heterologous reporter gene(15) , indicating that sequences
within the enhancer play an important role in the tissue-specific
expression of this gene. Here, we report the identification and
characterization of several transcription factor binding sites in the
BK6
enhancer, some of which may mediate its response to RA, TPA,
and hyperproliferation.
Figure 1:
Scheme of the 5`-upstream region of the
BK6 gene. The openbox represents the enhancer.
The sequence of this region is presented in Fig. 7. The
restriction sites are: B, BglII; H, HindIII; M, MnlI; S, Sau3A; X, XhoII; Xm, XmnI.
The lines above indicate the DNA fragments used in this study,
and the numbers their lengths (in bp). The BglII site
lies at the cap site.
Figure 7:
Sequence comparison of the 5`-upstream
regions of HK6 and BK6 genes. Sequences protected in the BK6
enhancer are boxed and labeled below. Sequences identified in
the HK6 promoter by other authors (29, 40) are underlined and labeled above. Tentative regulatory sequences
in the BK6
regulatory region are underlined and labeled
below. The TATA box, cap site, and ATG codon in the BK6
are shown
in boldfacetype. Restriction sites used to delimit
the BK6
enhancer fragments are indicated with arrows and initials (B, BglII; H, HindIII; M, MnlI; S, Sau3A; X, XhoII; Xm, XmnI).
Fragment 80 was able to produce a
single, specific retarded band. Fig. 2A shows that this
band is totally competed by an oligonucleotide containing the AP-1
element found to play an essential role in the BK5 gene
enhancer(20) , but not by an unrelated competitor. DNase I
footprints using this fragment gave a 23-nucleotide-long protected
region whose sequence is 5`-TGAGAGCATGACTAACCCATGAC-3`, between
positions -309 and -331 (Fig. 3A). This
region includes a TGACTAA sequence very similar to the consensus for
AP-1(22) . These results indicate a role for the Fos-Jun
complex in the BK6 gene regulation.
Figure 2:
Binding of nuclear factors to fragments of
the BK6 gene enhancer. 10 µg of BMGE+H nuclear extract
were used in each lane. Arrowheads indicate retarded bands. A, fragment 80. Lane1, no competitor DNA
added; lane2, addition of a 100-fold molar excess of
fragment PstI/XmnI from BK5, containing the AP-1 site
found in the enhancer of this gene(20) ; lane3, addition of a 100-fold molar excess of an unrelated
DNA fragment. B, fragment HX. Lane1, no
nuclear extract added; lane2, no competitor DNA
added; lane3, addition of a 100-fold molar excess of
unlabeled probe; lane4, addition of a 100-fold molar
excess of an unrelated DNA fragment. C, fragment MnlI. Lane1, no nuclear extract added; lane2, no competitor DNA added; lane3, addition of a 100-fold molar excess of unlabeled
probe; lane4, addition of a 100-fold molar excess of
an unrelated fragment.
Figure 3:
Identification of protein binding sites in
fragments 80 (A) and 95 (B) of the BK6 gene
enhancer by DNase I footprinting. End-labeled fragments 80 or 95 were
incubated with BMGE+H nuclear extracts and treated with DNase I as
described under ``Materials and Methods.'' Although only one
strand is shown, similar results were obtained with the opposite
strand. The protected sequences and their positions from the
translation start are shown. Lanes C, no nuclear extract
added; lanes 1 and 2, addition of different amounts
of BMGE+H nuclear extract; lane M, Maxam and Gilbert
sequence markers.
Fragment 95 also produced a specific retarded band when assayed with BMGE+H nuclear extracts (not shown). The nucleotide sequence of fragment 95 displays an AAACCAAA motif, which matches the consensus AAPuCCAAA (referred to as ``epidermal box'' in (23) ), that has been found preceding the TATA box in several genes expressed in epidermis, in particular keratins (23) and papillomavirus(24) . In spite of being located outside the enhancer, this sequence shows a footprinting protected region, 5`-ATGACTAAAGGAAACCA-3`, between positions -222 and -238 (Fig. 3B). This protected region includes a TGACTAA sequence, identical to the AP-1 element found in fragment 80, as well as the first 6 bp of the ``epidermal box,'' suggesting that this element could be a ``composite AP-1 site'' similar to others found in a number of genes (see, for instance, (25) ).
Fragment HX also
rendered a specific retarded band (Fig. 2B). DNase I
footprinting of this fragment showed a protected region,
5`-CCCCAATTCCCAG-3`, between positions -594 and -582 (Fig. 4A). Examination of the protected sequence and
the adjacent nucleotides allowed the identification of a TTCCCAGGC
sequence (most of which is contained in the footprint protected region)
that is highly homologous to the sequences that have been found to bind
transcription factor AP-2 in SV40 and in several keratin
genes(26, 27, 28, 29, 30) .
Although other putative protein binding sequences can also be found in
the protected region (see ``Discussion''), as AP-2 has been
involved in the regulation of several keratin genes, it is probable
that the protected element binds AP-2, although further work will be
necessary to confirm it. Interestingly, there is a TCTGCAGGC sequence,
between positions -135 and -143 (outside the enhancer),
highly homologous to the AP-2 also found upstream of the TATA box of
the human K14 keratin gene(27) . However, EMSA and DNase I
footprinting experiments with fragment 105, which includes this
sequence, were negative (not shown). These results suggest that the
AP-2-like sequence at -135 is not functional, which is supported
by the fact that deletion of this AP-2-like site does not alter the
proper expression pattern of a K6 promoter/lacZ construct
in transgenic mice. (
)In some footprint experiments with
fragment HX, we have also observed another protected, almost
palindromic region, 5`-GTGTCATGTCCC-3`, between nucleotides -604
and -615 (see Fig. 4A). We have not studied the
sequences protected in the HX fragment in greater detail due to the low
level of activity shown in CAT assays (see below).
Figure 4:
Identification of protein binding sites in
fragments HX (A) and MnlI (B) of the
BK6 gene enhancer by DNase I footprinting. End-labeled HX (A) or MnlI (B) fragments were incubated
with BMGE+H nuclear extracts and treated with DNase I as described
under ``Materials and Methods.'' Although only one strand is
shown, similar results were obtained with the opposite strand. The
protected sequences and their positions from the translation start are
shown at the right. LanesC, no extract
added; lanesE, addition of BMGE+H nuclear
extract; laneM, Maxam and Gilbert sequence
markers.
When we performed EMSA with fragment MnlI (which overlaps with fragments HX and 195, see Fig. 1), we found a major specific retarded band (Fig. 2C). DNase I footprinting experiments rendered two protected areas. One, spanning from nucleotide -582 to -594, is the same sequence protected at the equivalent position in fragment HX and therefore corresponds to the putative AP-2 site (not shown). The other protected region, 5`-AGAAGGACATGTTC-3`, extends from -536 to -549 and is shown in Fig. 4B. A detailed analysis of this sequence, in which a RARE is found, is presented below. This sequence was also the only protected region found in fragment 195 (not shown).
Given the complex tissue-specific
expression of K6, we also tested the pattern of protected regions in
footprinting experiments using nuclear extracts from PB cells, a murine
epidermal keratinocyte cell line which expresses K6. We obtained the
same protected sequences as described above for BMGE+H cells ( Fig. 5and data not shown), suggesting that similar transcription
factors bind the BK6 enhancer both in bovine mammary and murine
epidermal cell lines.
Figure 5:
DNase I footprinting of fragments of the
BK6 gene enhancer using PB nuclear extracts. End-labeled 80 (A) or MnlI (B) fragments were incubated
with PB nuclear extracts and treated with DNase I as described under
``Materials and Methods.'' The protected sequences and their
positions from the translation start are shown at the right. LanesC, no nuclear extract added. Lanes1 and 2, addition of different amounts of PB
nuclear extract.
Figure 6:
Analysis of the transcriptional activity
of fragments of the BK6 gene enhancer. A, BMGE+H
cells were either transfected (openbars) with
plasmids containing fragments HX, MnlI, or 80 cloned 5` to the
plasmid pBLCAT2, as well as with construct EXm-CAT2 (containing the
BK6
whole enhancer), or cotransfected (solidbars) with these same constructs and an expression
plasmid carrying the RAR
. 10
M RA was
added to the plates cotransfected with RAR
. 48 h after
transfection, cell extracts were collected and CAT activity assayed as
described under ``Materials and Methods.'' CAT activity is
represented as -fold stimulation over background (pBLCAT2 activity). B, Construct EXm-CAT2 (EXm) and a similar construct
in which the RARE has been deleted (-RARE) were
transfected (openbars) or cotransfected with an
expression plasmid carrying the RAR
(solidbars), as in A. 10
M RA was added to the plates cotransfected with RAR
. Both
constructs were similarly active in the absence of RA and
RAR
.
Response to vitamin D, RA, or
thyroid hormone is mediated by the orientation and spacing of
variations of the AGGTCA consensus(32, 33) . Such
elements can be found in the region protected in the MnlI
fragment, and also in the sequences preceding it, either in direct or
in palindromic repeats (Fig. 7), suggesting that this region
could be a target for regulation by retinoids. As it is known that K6
expression responds to RA treatment (13, 34, 35, 36, 37) , we
studied whether or not this response to RA was mediated by fragment MnlI. For this, we cotransfected BMGE+H cells with the MnlI-CAT2 plasmid and an expression plasmid coding for
RAR. Addition of 10
M RA to the
cotransfected plates increased CAT activity more than 10-fold (Fig. 6), indicating that the element found in the MnlI
fragment is indeed a RARE. Similar results were obtained with RAR
or RAR
(not shown). Cotransfection with RAR
, without addition
of RA, had only a slight activating effect in CAT activity. Similarly,
addition of 10
M RA to cells not
transfected with the RAR
plasmid increased CAT activity 2-fold
(not shown).
We also tested the response of other enhancer elements
to RA treatment. Fragments HX and 80 cloned in pBLCAT2 were
cotransfected with the RAR expression plasmid into BMGE+H
cells. Fig. 6A shows that, upon addition of
10
M RA, fragment HX remained inactive,
while fragment 80 lost most of its activity, probably due to AP-1
down-regulation produced by cross-talk interactions between Jun/Fos and
RAR(38) . However, the construct EXm-CAT2, bearing the whole
enhancer, was activated to a level similar to that obtained with
fragment MnlI. These results suggest that upon RA treatment,
the AP-1 element is inhibited and the transcriptional activity of the
enhancer is due fundamentally to the RARE located in the MnlI
fragment.
Since the only element in the BK6 enhancer that is
activated by RA seems to be the RARE, we eliminated it from the
enhancer by means of a two-step PCR (see ``Materials and
Methods''). The mutated and the wild-type enhancers were active at
similar levels when transfected in BMGE+H cells, demonstrating
that, as suggested above, the RARE does not play a significant role in
BK6
regulation under normal conditions. However, when
cotransfected with RAR
, in contrast to the wild-type enhancer, the
mutant enhancer failed to be stimulated by RA to appreciable levels (Fig. 6B), suggesting that the RARE found in the MnlI fragment mediates the RA induction of the enhancer.
In addition to the identified
elements, a number of potential binding sequences can be found in the
BK6 5`-upstream region (Fig. 7). For instance, there is a
palindrome TTCAGTGAA at position -308, immediately downstream of
the AP-1 site, and a duplication of the CATACTT motif at position
-208, which coincides with the palindrome sequence of HK6 known
to bind a nuclear factor(39) . Finally, at position -149,
there is a stretch of 28 nucleotides perfectly conserved between the
two genes. The significance of these sequences is at present unknown.
Keratin genes, due to their complex pattern of epithelial
tissue expression, constitute very attractive systems for the study of
gene regulation. K6 regulation seems to be particularly complex (see
Introduction). We have identified in BK6 a tissue-specific
enhancer located between positions -180 and -605 from the
cap site(15) , which is necessary to provide a correct
tissue-specific as well as inducible expression of a
reporter(44) . To understand BK6
gene regulation, we have
studied the nuclear transcription factors binding to the region
comprising this enhancer and the proximal promoter zone. Some of the
sequences found (two AP-1, one of them composite, and a RARE) and their
interactions permit an explanation, at least in part, of the induction
of this gene in hyperproliferative situations and the complex behavior
of K6 when epidermal keratinocytes are treated with RA.
AP-1 is a
regulatory element that has been found in an increasing number of
keratin genes: HK18(45) , HK1(46) , BK5(20) ,
HK19(47) , HK6 (39) , and here, in BK6. However,
the AP-1 sequence, found once in HK6 (39) and twice in
BK6
, is not the perfect palindromic consensus
TGACTCA(22) , but TGACTAA. Since this sequence is identical to
the AP-1 sequences found in the upstream regulatory regions of certain
papillomaviruses (48, 49, 50) that are active
in the suprabasal layers of stratified epithelia where K6 is also
expressed, it is tempting to speculate that this particular AP-1
variant may be especially relevant mediating Fos/Jun action in these
types of suprabasal cells. It has been found that different AP-1
sequences bind preferentially to different Jun and Fos
proteins(51, 52) , and perhaps the combination of
Jun/Fos that exists in these suprabasal layers binds better to the
variant form TGACTAA. The two AP-1 elements that we have found are very
likely involved in the suprabasal K6 induction by TPA and
hyperproliferative stimuli. Another similarity between HPV18 and
BK6
is that in HPV18, tissue-specific expression is mediated by a
cooperation between AP-1 and a close factor (KRF-1), and Oct-1 blocks
this cooperation by binding to a KRF-1 overlapping binding
site(50) . Strikingly, in the BK6
enhancer, and at the
same distance from the AP-1 site as in HPV18 (although in an inverted
orientation; see (48) for a map of HPV18 regulatory elements),
there is an Oct-1 sequence and a palindrome lies between these two
elements (see Fig. 7). Although we have not detected any protein
binding to these elements, they could play a role in BK6
regulation. In this respect, Oct-related proteins have been recently
identified as regulators of keratin gene expression (53, 54) .
It is difficult to ascertain the
implication of AP-2 in BK6 gene regulation. Although the sequence
protected in the HX fragment fits the AP-2 consensus well (26) and agrees well with AP-2 sequences found in several
keratin genes (see (27) and (55) ), some other protein
binding sequences can be found in this region, such as a CCAAT box, as
well as binding sites for H-APF-1 (TTYCCAG, (56) ) and the
adenovirus transcription factor E4F (AATTCCCA, (57) ). However,
the fact that none of these factors has been related previously to
keratins, together with the repeated involvement of AP-2 in keratin
regulation(27, 28, 29, 30) ,
suggests that this sequence is a binding site for AP-2, although more
work will be necessary to establish this point.
Retinoic acid exerts
profound and complex effects on epidermal differentiation(58) .
The action of RA on keratin expression is also complex, and probably
dependent on concentration and environmental conditions. Thus, the
K1/K10, K5/K14, and K6/K16 pairs have been reported to be
down-regulated by 10M RA in vitro at the transcriptional level(36, 59) . However,
not only is the synthesis of the first two pairs not decreased in
RA-treated skin in vivo, the K6/K16 pair is induced both in
human and rodent
skin(13, 14, 35, 37) . An important
result of our work has been the identification of a RARE in the
BK6
enhancer, which is strongly stimulated in vitro by
RA, and whose deletion eliminates the response of the enhancer to RA.
These results are at variance with those of Tomic-Canic et al. (60), who found an element responding negatively to RA in the HK14
promoter. However, since there is no significant identity between the
BK6
and HK14 RAREs, these two elements could be functionally
different.
Our results explain the behavior of the BK6 enhancer
in a relatively straightforward manner. On the one hand, under normal
culture conditions the activity of the enhancer seems to be due mainly
to the AP-1 element, which accounts for 70% of the total enhancer
activity. There is no apparent synergism between AP-1 and the other
identified elements (AP-2, RARE), since the activity of the total
enhancer is the sum of the activities of the component elements (see Fig. 6). On the other hand, the presence of RAR
and RA
induces a positive response of the RARE element. At the same time, the
activity of fragment 80, containing the AP-1 element, is drastically
diminished due to negative cross-talk between the AP-1 and RAR, as has
been described in other systems(38) , and even in
HPV18(61) . The effect of RA on the enhancer may be interpreted
as a combination of the two effects, AP-1 inhibition and RARE
stimulation, with the net effect of enhancer activation in BMGE+H
cells. The finding in the BK6
enhancer of elements that are either
activated (RARE) or inhibited (AP-1) by RA could help explain the
complex and sometimes apparently contradictory response of K6 to RA
both in vivo and in vitro. The induction or
inhibition of this keratin may be the result of a delicate balance
between isoforms of Fos and Jun, type and amount of RARs, and the
effective concentration of RA. These elements would depend on cell type
and treatment conditions, explaining, for instance, why K6 has been
shown to be induced in vivo and repressed in vitro by
RA(13, 14, 35, 36, 37) ,
why the degree of RA-induced K6 inhibition is much stronger in SCC13
cells than in primary epidermal keratinocyte
cultures(34, 36) , or why different retinoids may
stimulate or inhibit K6 expression(62) . However, when
considering K6 induction by RA or hyperproliferative conditions, one
should consider that K6 is a minigene family. So far, three K6 variants
have been identified in bovids and two in humans, and different
isoforms could be regulated in different ways.