(Received for publication, August 2, 1995; and in revised form, November 17, 1995)
From the
In this study, we describe the isolation and characterization of
a cDNA clone C12 that encodes a new member of the cornifin/small
proline-rich protein (spr) family, which we have named cornifin .
C12 encodes a 1.1-kilobase pair mRNA and a 24.3-kDa cytosolic protein
with a high proline content (19%). Its total amino acid sequence
exhibits a 37-66% identity while the first 30 amino acids at the
amino terminus are 87% identical to that of members of the cornifin
family. At its carboxyl terminus, cornifin
contains 21 tandem
repeats of an octapeptide. Cornifin
expression is restricted to
several squamous epithelia. It is highly expressed in esophagus,
tongue, and oral mucosa but, in contrast to cornifin
, is not
detectable in the epidermis. Both retinoic acid and a retinoid
selective for the nuclear retinoic acid receptors were very potent
suppressors of cornifin
expression while an analog selective for
the nuclear retinoid X receptors was much less effective, suggesting
that a specific retinoid signaling pathway is involved in this
suppression. Cornifin
can function through some of its Gln
residues as an amine acceptor in transglutaminase-catalyzed
cross-linking reactions. These results indicate that cornifin
functions as a cross-linked envelope precursor.
Squamous differentiation is a multistage process that is
accompanied by irreversible growth arrest and expression of squamous
cell-specific genes(1, 2, 3, 4) .
The formation of the cross-linked envelope is a characteristic feature
of squamous differentiation in many tissues (5, 6) .
This structure consists of a layer of covalently cross-linked protein
deposited just beneath the plasma
membrane(6, 7, 8, 9) . These
linkages are catalyzed by transglutaminases, enzymes that carry out the
formation of -(
-glutamyl)lysine bonds between precursor
proteins (7, 8, 9, 10, 11, 12) .
The formation of the cross-linked envelope is believed to occur in
several stages and to involve multiple membrane-associated and
cytosolic precursor proteins, including involucrin, cornifin/small
proline-rich protein (spr), (
)and loricrin (11) .
The first described envelope precursor, involucrin, is a glutamine-rich
protein induced early during squamous differentiation (9, 13, 14) . Loricrin, a glycine-rich
precursor protein, is induced at later stages of differentiation than
involucrin and cornifin and appears to be the major constituent of the
mature cross-linked envelope (15, 16) . Cornifins and
sprs are a family of related envelope precursor proteins(17, 18, 19, 20, 21, 22) which
contain a highly repeated octapeptide (nonapeptide for spr2) at their
carboxyl terminus and a high percentage of proline. Cornifin
has
been reported to be an excellent substrate for epidermal (type I)
transglutaminase and has also been shown to be assembled into the
cross-linked envelope(20) .
In this study, we describe the
characterization of C12, a cDNA clone isolated from a library prepared
from poly(A) RNA of squamous-differentiated rabbit
tracheal epithelial (RbTE) cells. This clone represents an mRNA that is
present at high abundance in squamous-differentiated cells but not in
undifferentiated RbTE cells. Based on its amino acid sequence homology
with the previously described cornifin/sprs, C12 is a new member of the
cornifin/spr
family(17, 18, 19, 20, 21, 22) .
We have named the previously described cornifin (SQ37)(20) ,
cornifin
, and the protein encoded by C12, cornifin
. We show
that cornifin
functions as a substrate for transglutaminase type
I indicating that it can also function as a cross-linked envelope
precursor. The fact that the sequence of the tandem repeats and pattern
of tissue-specific expression are different between the two cornifins
may suggest distinct roles for specific cornifins, perhaps in
determining the physical properties of the cross-linked envelope.
Differential screening of a cDNA library prepared from
poly(A) RNA isolated from squamous-differentiated RbTE
cells yielded several cDNA clones encoding mRNAs that were abundantly
expressed in squamous differentiated RbTE cells but not in
undifferentiated cells(17) . In this study, we describe the
characterization of one of these cDNA clones named C12 and its
derivative C12-3. These cDNAs contain inserts encoding overlapping 3`-
and 5`-fragments of a novel squamous cell-specific mRNA. These inserts
were sequenced in both directions; the cDNA sequence is shown in Fig. 1. A putative initiation codon was present 27 bases from
the 5`-end of the cDNA. The open reading frame (ORF) terminates with a
stop codon at nucleotide 720. A polyadenylation signal (AATAAA) was
found 308 nucleotides further from the stop codon. Based on the deduced
amino acid sequence, the mRNA encodes a hydrophilic 24.3-kDa protein
with an estimated pI of 8 (Fig. 1).
Figure 1:
Nucleotide and deduced amino acid
sequence of C12 (cornifin ). The putative initiation codon (ATG),
termination codon (TAG), and the polyadenylation signal (AATAAA) are
indicated in boldface type. The amino acid sequence is shown
in the single-letter code. The underlined amino acid sequence
represents the peptide C12-PEPB used to raise
antibodies.
Data base searching (GCG
FastA on the combined nucleic acid data base) with the ORF of C12
revealed substantial similarity to sprs and cornifin
(18, 19, 20) . The DNA coding sequence
of C12 exhibited a 51% identity with that of cornifin
(20) . The amino acid sequence of C12 was 49, 57, 37, and
66% identical to cornifin
, spr1, 2, and 3,
respectively(18, 19, 20) . The first 30 amino
acids at the amino terminus were the most highly conserved (87%)
between C12, cornifin
and the spr's (Fig. 2A). Like cornifin
/spr1 and spr3, C12 has a
high proline content (19%) and contains a highly repeated octapeptide
at the carboxyl terminus. However, these repeats deviate substantially
from the consensus repeat sequences of cornifin
/spr1 and spr3 (Fig. 2B). Moreover, the octapeptide repeats in C12
were not as highly conserved between one another as those in cornifin
or the sprs. Four subclasses of octapeptides could be identified
in C12 exhibiting a 40-60% identity between one another and a
25-50% and 50-87% identity with sequences in cornifin
and spr3, respectively (Fig. 2B). These octapeptides
each were duplicated two to six times. These results indicate that C12
encodes a protein that is distinct but closely related to cornifin
and sprs. This protein was named cornifin
.
Figure 2:
Comparison of the amino acid sequence of
cornifins and sprs. A, comparison of the first 30 amino acids
at the amino terminus. The amino acids shown in bold vary from the
amino acid sequence of cornifin (C12). SPR1, 2, and 3 are the
sequences of the human small proline-rich
proteins(18, 19) . B, comparison of the
different octapeptides present in the tandem repeats of cornifins
and
, and spr3. The arrows line up the repeats that are
identical or exhibit a high degree of homology. Two (A and B) such repeats can be identified in cornifin
, four (C, D, E, and F) in cornifin
and two in spr3 (G and H).
We next
examined by Northern blot analysis and in situ hybridization
the tissue-specific expression of cornifin mRNA and its
regulation during squamous differentiation. Northern blot analysis
showed that C12 represents a 1.1-kb mRNA that is induced when RbTE
cells undergo squamous cell differentiation (Fig. 3A).
Squamous differentiated RbTE cells express more than 50-fold higher
levels of cornifin
mRNA than undifferentiated cells. Furthermore,
Northern blot analysis of RNA from different rabbit tissues showed that
cornifin
mRNA was highly expressed in esophagus and tongue and
present at low levels in lip, but was not detectable in kidney, liver,
brain, or testis (Fig. 3B). Although expression of
cornifin
is highly restricted to several squamous differentiating
tissues, it was, in contrast to cornifin
, undetectable in rabbit
skin. Similar observations were obtained with human tissues. Cornifin
was highly expressed in human esophagus but was undetectable in
epidermis and squamous-differentiated human epidermal keratinocytes in
culture (not shown). Thus, cornifin
, rather then being a general
marker for squamous differentiation, is expressed only in certain
squamous tissues.
Figure 3:
Expression of C12 mRNA in squamous
differentiating cells. Total RNA (30 µg) prepared from RbTE cells
and various rabbit tissues were fractionated, blotted to Nytran, and
hybridized to P-labeled probes for C12 or glyceraldehyde
3-phosphate dehydrogenase (GPDH). A, RNA from
undifferentiated and squamous differentiated RbTE cells. B,
RNA from various rabbit tissues.
The conclusion that expression of cornifin
is induced during squamous cell differentiation was further confirmed
in studies examining the localization of cornifin
transcripts by in situ hybridization. Expression of cornifin
mRNA was
restricted to the suprabasal layers of the rabbit esophageal epithelium (Fig. 4) and other squamous epithelia such as the tongue and the
oral mucosa (not shown). This pattern of expression is very similar to
those reported previously for cornifin
and transglutaminase type
I (21, 31) and confirms that cornifin
expression
is associated with squamous differentiation. However, once again
cornifin
mRNA was not detectable in the epidermis (not shown).
Figure 4:
Localization of C12 mRNA in rabbit
esophagus by in situ hybridization. In situ hybridization on sections of rabbit esophagus was carried out as
described under ``Experimental Procedures'' using S-labeled sense (A and C) and antisense (B and D) C12 probes. A and B,
bright field; C and D, dark field
exposure.
To analyze the expression of cornifin at the protein level, an
antiserum was raised against the synthetic peptide C12-PEPB (Fig. 1A). The antiserum recognized a major protein in
total protein extracts of squamous differentiated RbTE cells that
migrated at an apparent molecular mass of about 32 kDa (Fig. 5A). The latter is higher than the predicted
molecular mass, as has also been observed for cornifin
(20) . In several experiments the antiserum reacted weakly
with another protein migrating at a slightly lower molecular mass (28
kDa). This smaller immunoreactive protein may have been derived from
cornifin
by proteolytic digestion. The specificity of the
immunoreactivity was shown by competitive blocking of the
protein-antiserum interaction with the homologous peptide but not with
a heterologous peptide (Fig. 5A). In addition,
preimmune serum did not react with any protein in extracts from
squamous differentiated RbTE cells (not shown). Fig. 5, B and C, shows the induction of cornifin
in relation
to the onset of squamous differentiation in RbTE cells, which is
induced when cultures reach confluence (at day 8 and
9)(1, 11) . Cornifin
was detectable only in
confluent, squamous-differentiated cultures but not in logarithmic
cultures containing undifferentiated cells. Cornifin
was
increased more than 50-fold when cultures of RbTE cells reached
confluence.
Figure 5:
Immunoblot analysis of cornifin
protein expression. Proteins from undifferentiated and
squamous-differentiated RbTE cells were examined by immunoblot analysis
using C12-PEPB-Ab antiserum. A, immunoblot analysis of total
cellular protein from squamous-differentiated cells (lane 1),
in the presence of the homologous peptide (lane 2), or
heterologous peptide (lane 3). B and C,
induction of cornifin
protein during differentiation of RbTE
cells. RbTE cells were plated at 5
10
cells/60-mm
dish and at the times indicated cells were collected for the
determination of cell number (B) and cornifin
protein (C) by immunoblot analysis. The molecular mass of protein
markers (kDa) is indicated on the right.
In agreement with the results obtained by Northern
blotting and in situ hybridization, immunoblot and
immunohistochemical analysis indicated that cornifin expression
is associated with squamous differentiation in several, but not all,
squamous tissues. Cornifin
was detectable in extracts from rabbit
tongue, esophagus, and oral mucosa but undetectable in skin, trachea.
muscle, and liver (Fig. 6). The localization of cornifin
was analyzed by immunohistochemical staining and compared with that of
cornifin
and involucrin. In sections of esophageal epithelium,
immunoreactivity with the C12-PEPB antiserum is restricted to the
suprabasal layers (Fig. 7A). Although the staining
pattern for cornifin
is very similar to that for involucrin, it
appears that the induction of involucrin occurs somewhat earlier (Fig. 7C). The immunoreactivity for cornifin
occurs in layers that are closer to the lumen than those stained for
involucrin and cornifin
. These results suggest that both cornifin
and involucrin are induced at an earlier stage of differentiation
than cornifin
.
Figure 6:
Expression of cornifin in different
rabbit tissues. Protein extracts from the epithelium of the rabbit
tongue (1), esophagus (2), oral mucosa (3),
skin (4), trachea (5), muscle (6), and liver (7) were examined by immunoblot analysis using anti-C12-PEPB
antiserum. The molecular mass (kDa) of protein markers is indicated on
the left.
Figure 7:
Comparison of the expression of cornifin
and
and involucrin in the esophagus by
immunohistochemistry. Sections of human esophagus were analyzed by
immunohistochemistry using rabbit antisera against A, cornifin
; B, cornifin
; C, involucrin; and D, rabbit preimmune serum.
Fractionation of the cellular lysates showed
that cornifin was associated predominantly with the soluble
fraction suggesting that it is a cytosolic protein (Fig. 8A). Since cornifin
is related to the
cross-linked envelope precursor cornifin
(20) , we
determined whether it can also serve as a substrate for
transglutaminase type I which catalyzes the formation of
(
-glutamyl)lysine isopeptide bonds between cross-linked
envelope precursors(7) . We first examined whether cornifin
becomes cross-linked when cells are treated with calcium
ionophore. Such an exposure increases the intracellular Ca
level which activates transglutaminase type I leading to
subsequent cross-linking of envelope
precursors(10, 20) . Fig. 8A shows
that the reactivity of cornifin
with the antibody against
C12-PEPB is abrogated after the cells are treated with the calcium
ionophore Ro 2-2985 in agreement with the concept that it becomes
cross-linked into high molecular weight aggregates. Varying degrees of
epitope masking have been observed previously during cross-linking of
envelope precursors (20) and is probably responsible for the
loss of immunoreactivity of the cross-linked C12 protein as well.
Figure 8:
Transglutaminase-induced cross-linking of
cornifin protein. A, total cellular protein (T), the soluble protein fraction (S) and the
particulate protein fraction (P) from untreated and
Ca
-ionophore-treated squamous differentiated RbTE
cells were examined by immunoblot analysis with C12-PEPB-Ab. B, identification of proteins in differentiated RbTE cells
that are covalently cross-linked with dansylcadaverine. Total cellular
extracts were incubated in the presence of dansylcadaverine for 0, 2,
5, and 10 min (lanes 1, 2, 3, and 4). Samples were then examined by immunoblot analysis using E7
monoclonal antibody (DANS) (29) or antisera against
C12-PEPB or SQ37A-Ab (20) for the presence of dansylated
proteins, cornifins
and cornifin
,
respectively.
In
order to confirm cross-linking of cornifin , dansylcadaverine was
supplied as an amine donor in in vitro cross-linking
reactions(10, 20) . As shown in Fig. 8B, two major proteins of 21 and 32 kDa in crude
extracts prepared from differentiated RbTE cells were covalently linked
to dansylcadaverine in a time-dependent manner. The smaller protein
comigrated with cornifin
(20) , the larger one with
cornifin
.
The expression of many squamous cell-specific genes
have been reported to be down-regulated by
retinoids(1, 2) . Therefore, we examined the effect of
several retinoids on the expression of cornifin . As shown in Fig. 9A, 10
M retinoic acid
totally suppressed the induction of cornifin
mRNA. To obtain more
insight in the signaling pathway involved in this retinoid action, the
effect of two retinoid receptor selective retinoids was determined.
Nanomolar concentrations the RAR-selective retinoid SRI 6751-84 were
able to suppress the expression of both cornifin
and
(SQ37)
very effectively, whereas the RXR-selective retinoid SRI-11217 was much
less potent. Cornifin
may be slightly less sensitive to retinoids
than cornifin
. The repression of cornifin
by retinoids was
also observed at the level of the protein (Fig. 10). The
retinoid SR11302, which does not induce RAR- or RXR-dependent
transactivation but inhibits AP1-dependent
transactivation(24) , had no effect on the expression of
cornifin
(not shown).
Figure 9:
Effect of RAR- and RXR-selective retinoids
on cornifin and
mRNA expression. RbTE cells were grown to
confluence and then treated with RA, RAR-selective (RAR
) or RXR-selective (RXR
) retinoids at the concentrations
indicated. Five days later cells were collected and total RNA isolated.
RNA (30 µg) was analyzed by Northern blot analysis using
P-labeled probes for C12 (A), cornifin
(SQ37) (B), and GPDH.
Figure 10:
Suppression of cornifin protein by
the RAR-selective retinoid SRI 6751-84. Subconfluent cultures of RbTE
cells were treated with the RAR-selective retinoid SRI 6751-84 (RAR
) at the indicated concentrations
and four days later cells were collected and examined by immunoblot
analysis using anti-C12-PEPB antiserum.
In this study, we describe the isolation and characterization
of a novel cross-linked envelope precursor which is a new member of the
cornifin/spr family. This protein was named cornifin . The lines
of evidence supporting this classification include the presence of a
highly conserved amino-terminal region characteristic of cross-linked
envelope precursors and of a characteristic, highly repeated
octapeptide. In addition, the strong association of its expression with
squamous differentiation and its ability to serve as a substrate in
transglutaminase-catalyzed cross-linking reactions. The predicted amino
acid sequence of cornifin
is 49% identical to that of the
previously reported cornifin, referred to now as cornifin
(20) , and exhibits a 57, 37, and 66% identity to that of
spr1, 2, and 3, respectively(18, 19) . The sequence of
the 30 amino acids at the amino-terminal region are remarkably well
conserved, 87% between rabbit cornifin
and cornifin
. It is
also well conserved across species (Fig. 2). Interestingly, this
sequence also shows considerable homology to the amino-terminal region
of two other cross-linked envelope precursors, involucrin and
loricrin(13, 15, 16) . Although one could
expect this highly conserved region to be derived by the duplication of
a single exon, no intron was found at the borders of this
region(19) . (
)
As for cornifins and sprs,
cornifin is rich in amino acids that can disrupt protein
secondary structure. Cornifin
has a proline content of 19%
compared to 31% for cornifin
and 22% for spr3(19, 20) and a glycine content of 8% versus 0 and 9% for
the other two, respectively. The percentage of glutamine, lysine, and
cysteine in cornifin
(8.6, 6.9, and 3.0%, respectively) is much
lower than that in cornifin
(20, 13, and 11%, respectively).
Cornifin contains a highly repeated octapeptide at its
carboxyl terminus as do cornifin
, spr1 and 3. Cornifin
contains 12 repetitions of the highly conserved consensus sequence
EPCQPKVP, whereas cornifin
contains 21 octapeptide repeats. These
sequences are not as highly conserved as those in cornifin
and
spr3. The cornifin
repeat sequences fall into four subclasses:
ESGCTSVP, QP(G/S)YTKVP, GPGYPTVP, and GSGYSV(V/I)P which are repeated,
respectively, five, five, two, and three times (Fig. 2B). These octapeptides can be viewed as being
organized in groups of three as repeats of a 24-amino acid sequence.
During evolution, the cornifin
sequence may have arisen from
duplications of the octapeptide followed first by mutations leading to
a diversion in amino acid composition between the octapeptides and
subsequent duplications of the 24-amino acid sequence. The sequence and
organization of the repeats in cornifin
deviate substantially
from those found in cornifin
and spr3, yet still one-third of the
amino acids in this region tend to disrupt conventional secondary
structure. The repeat sequences exhibit a 25-50% and 50-87%
identity with sequences in cornifin
and spr3. No similarity in
the sequence of these repeats were observed with those found in other
squamous cell marker genes such as involucrin, loricrin, and
filaggrin(13, 15, 16, 31) .
As
cornifin , cornifin
can function as a substrate for
transglutaminase type I(20) . This was indicated by treatment
of RbTE cells with calcium ionophore Ro 2-2985 which results in the
activation of transglutaminase type I and the disappearance of
immunoreactive cornifin
when it becomes cross-linked and
associated with the cross-linked envelope. In addition, labeling of
proteins with dansylcadaverine revealed two major labeled proteins of
21 and 32 kDa. Previous studies identified the 21-kDa band as cornifin
and showed that the larger 32-kDa protein did not
immunoprecipitate with anti-cornifin
antibodies(20) .
This protein comigrates at the same position as cornifin
,
suggesting that this dansylated protein is cornifin
.
The
expression of cornifin is associated with squamous
differentiation. This is demonstrated by Northern blot, in situ hybridization, and immunohistochemical analyses showing that the
presence of cornifin
mRNA and protein was restricted to squamous
epithelia and limited to the suprabasal layers of the squamous
epithelium. The tissue-specific expression of cornifin
appears to
be more restricted than that of cornifin
. In contrast to cornifin
, cornifin
was expressed in neither rabbit nor human skin
nor in cultured NHEK cells; however, cornifin
and
each were
abundantly expressed in both rabbit and human oral mucosa, esophagus,
and tongue ( Fig. 4and Fig. 6)(20, 21) . (
)The differential expression of cornifin
and
may through alterations in the composition of the cross-linked envelope
determine different physical properties of that structure as may be
required in different tissues. Immunohistochemical analyses indicate
that cornifin
and
, involucrin and loricrin are induced at
different points during squamous
differentiation(14, 15, 21) . This sequential
induction supports the hypothesis that the formation of the
cross-linked envelope is a multistep process(20) . As proposed
previously, involucrin and cornifin may form a scaffold upon which
loricrin and perhaps other cross-linked envelope precursors are
assembled.
The induction of cornifin protein and mRNA is
suppressed by retinoic acid. Retinoids have been shown to mediate their
action on gene expression through specific nuclear retinoid receptors,
RARs and RXRs (reviewed in Giguere(32) ). To examine what
retinoic acid signaling pathway is involved in this suppression, the
action of SRI 6751-84 and SRI-11217, an RAR- and an RXR-selective
retinoid, respectively, and of SR11302, a retinoid that exhibits
anti-AP-1 activity but that is unable to induce transactivation through
the retinoid response elements, RARE or RXRE(25) , were
studied. In contrast to the RXR-selective retinoid, the RAR-selective
retinoid was very effective in suppressing the expression of cornifin
mRNA while SR11302 had no effect. These observations suggest that
the suppression of C12 is mediated through activation of RARs rather
than RXRs and appears not to require the anti-AP1 activity of
retinoids. Characterization of the DNA elements involved in the
up-regulation and the retinoid-mediated repression of this gene has to
await the isolation of the promoter region of cornifin
.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U40631[GenBank].