(Received for publication, November 20, 1995; and in revised form, January 22, 1996)
From the
Transglutaminase 1 (TGase 1) is expressed during the terminal
differentiation of keratinized squamous epithelium to form cornified
cell envelope in differentiated keratinocytes by the
-(
-glutamyl) cross-linking reaction. The gene for human TGase
1 is responsible for autosomal recessive lamellar ichthyosis, a severe
hereditary keratinizing disorder of the skin. We examined the
transcriptional activity of the gene in FRSK, rat keratinocytic cells,
transfected with the luciferase reporter gene under control of the 5`
upstream region of human TGase 1 gene. Transfection of the reporter
gene with an expression vector for the
isoform of novel protein
kinase C (nPKC
), as well as exposure to
12-O-tetradecanoylphorbol-13-acetate, markedly increased the
luciferase activity in FRSK, but not in HT-1080 fibrosarcoma cells,
although exogenous nPKC
was expressed in both. The induction was
suppressed by deleting the TGase 1 upstream sequence from -95 to
-67 and by deleting the kinase domain from exogenous nPKC
.
In comparison with other PKC isoforms, nPKC
most effectively
induced the luciferase activity. We suggest that nPKC
, an
epithelium-specific isoform of PKC, mediates the activation of the
TGase 1 transcription.
Terminal differentiation is a highly organized biological
process that involves the programmed death of cells(1) . The
epidermis covering the most outer layer of the skin is maintained by a
dynamic equilibrium of renewal with terminal differentiation of
keratinocytes, the major cell lineage of the epidermis. This process,
keratinization, proceeds with the ordered expression of genes in
keratinocytes and their morphological changes. Transglutaminase 1
(TGase 1), ()a member of the transglutaminase family (EC
2.3.2.13), is expressed during
keratinization(2, 3, 4) . The enzyme
catalyzes the
-(
-glutamyl) lysine cross-linking reaction of
the proteins, such as involucrin(5) ,
cystatin-
(6) , loricrin, small proline-rich proteins, and
elafin(7) , and forms the cornified cell envelope on the inner
surface of the differentiated keratinocytes (4) . This
insoluble structure maintains the internal milieu and protects the body
against the chemical, mechanical, and biological invasion. The
importance of this enzyme in the morphogenesis and barrier function of
the skin has been shown also by discovery of its mutation in autosomal
recessive lamellar ichthyosis(8, 9, 10) ,
which is a hereditary keratinization disorder characterized by
collodion baby at birth and later, severe ichthyosis with large
brownish plate-like desquamations and epidermal hyperkeratosis,
ectropion, eclabium, and scarring alopecia.
The gene for human TGase
1 is located on 14q11.2 (11, 12) and encodes a 92-kDa
protein consisting of 816 amino acid residues (13, 14, 15) . In the epidermis of the skin,
TGase 1 gene is expressed mainly in the subcorneal granular
layers(16, 17, 18) . The enzyme activity of
transglutaminase is enhanced by treating primary mouse epidermal cells
with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator
of protein kinase C(19) . In cultured epidermal keratinocytes
and tracheal epithelial cells, the level of TGase 1 mRNA is increased
also by TPA (20, 21) as well as
Ca(22, 23, 24) ,
ganglioside GQ
(24) , and
interferon-
(25) . Besides the effect of TPA, the critical
function of PKC in the regulation of TGase 1 gene expression has been
suggested by studies using several PKC
inhibitors(20, 26) . However, there is no direct
evidence indicating that PKC mediates the signal for the
transcriptional activation of TGase 1.
PKC was initially
characterized as a serine/threonine-type protein kinase, the activity
of which is dependent on Ca, phospholipids, and
diacylglycerol(27, 28, 29) . Subsequently,
various isoforms of the enzyme have been identified(30) . PKC
is now classified into three major subgroups, Ca
-,
phospholipid-, and diacylglycerol-dependent classical PKCs (cPKC)
(
,
I,
II, and
), Ca
-independent
novel PKCs (nPKC) (
,
,
, and
) and
Ca
- and diacylglycerol-independent atypical PKC
(aPKC) (
and
/
). In addition, the µ isoform, a
membrane-bound PKC, has been identified(31) . They differ in
terms of tissue distribution and are involved in a variety of
specialized functions of the tissues. One nPKC, called nPKC
(PKC-L), is epithelium-specific(32, 33) . Although
there are also other PKC isoforms, such as cPKC
and nPKC
, in
the epidermis of the skin (34) , the localization of nPKC
in the subcorneal granular layer is quite similar to that of TGase 1
mRNA(16, 17) , suggesting that it mediates the
activation signal for TGase 1 gene during the terminal differentiation
of epidermal keratinocytes. Understanding the unique signal
transduction for TGase 1 gene regulation should provide new insight
into the molecular mechanism involved in the complex biological process
of keratinization. Therefore, we analyzed the role of nPKC
in the
transcriptional activation of TGase 1 gene expression.
Figure 1: The TGase 1-luciferase reporter genes. Deletions of the human TGase 1 5` flanking region from -819 to -49 were generated by PCR and inserted into the SmaI site of PGV-B, a basic luciferase reporter vector. PGV-C is a constitutive expression vector for luciferase driven by SV40 promoter and enhancer. Arrows indicate PCR primers used to synthesize the deletion mutants. Upper primers are as follows: pd-819, aattccaggttggagaaaagg; pd-95, cccatttcccgcccagaggcc; pd-66, tctctccgccccctacagcag; pd-49, gcagtttggcccctccctccc. The lower primer is tcacttaccaggtctgtccct. This primer was also used to prepare the deletion fragment pd-20, by annealing it with its sense oligonucleotide. E, EcoRI; P, PvuII; B, BamHI; H, HindIII; Luc, luciferase gene; SV40 box, SV40 enhancer; P box, SV40 promoter.
Figure 2:
The transcriptional activation of the
human TGase 1 gene by TPA and nPKC. A, the effect of TPA
on the transcriptional activity of human TGase 1. The TGase
1-luciferase reporter genes were transfected into FRSK, cultured rat
keratinocytes. After a 2-day incubation, the cells were exposed or not
to 1 nM TPA for 4 h, and then the luciferase activity was
determined, as described under ``Experimental Procedures.'' B, the effect nPKC
on the transcriptional activity of
human TGase 1. SRD
, an expression plasmid for nPKC
, was
co-transfected with the reporter genes into FRSK cells; 2 days later,
the luciferase activity was determined. Data represent the means of
duplicate experiments normalized to
-galactosidase activity. PGV-B
and PGV-C are negative and positive control reporter plasmids,
respectively.
Figure 3:
The comparison of the effects of
nPKC on the transcriptional activation of human TGase 1 gene
between FRSK cells and HT-1080 cells. A reporter gene, pd-95, was
co-transfected with the indicated amounts of SRD
into FRSK cells (A) or into HT-1080 fibrosarcoma cells (B), after
which the luciferase activity was determined, as described under
``Experimental Procedures.'' pL27 is an expression vector for
human nPKC
(PKC-L). Data represent the means of duplicate
experiments normalized to
-galactosidase
activity.
In contrast to FRSK cells, TPA fails to
activate TGase 1 transcription in HT-1080, a human fibrosarcoma cell
line, even when a reporter plasmid containing -819 upstream
sequences is transfected(38) . When pd-95 was co-transfected
with SRD from 2.5 to 25 fmol into HT-1080 cells, the induction of
luciferase activity remained at low levels (Fig. 3B),
indicating that the machinery that transduces the signal elicited by
nPKC
is defective in HT-1080 cells.
Figure 4:
The effect of mutant SRD on the
human TGase 1 transcriptional activity in FRSK cells. A reporter gene
pd-95 was co-transfected into FRSK cells with 12.5 fmol of either
SRD
or SRD
RD, a mutant SRD
with deletion in the
sequence encoding the kinase domain of nPKC
, as described under
``Experimental Procedures.'' After 2 days of incubation, the
cells were incubated with or without 1 nM TPA for 4 h, and
then luciferase activity was determined. Data represent the means of
duplicate experiments normalized to
-galactosidase
activity.
Figure 5:
PKC isoform expression in FRSK and HT-1080
cells transfected with PKC expression vectors. Seven micrograms of
proteins extracted from the cells transfected with SRD vector or with
various amounts of PKC expression vectors were separated by SDS-PAGE,
transferred to a nylon membrane, and reacted with antiserum against
nPKC (A and B), cPKC
(C), and
nPKC
(D), as described under ``Experimental
Procedures.'' Lane 1, no transfection; lane 2,
transfection with 500 fmol of SRD vector; lanes 3-6,
transfection with 25, 125, 250, and 500 fmol of PKC expression vectors,
respectively; lane 7, transfection with 500 fmol of pL27
encoding human nPKC
.
Figure 6:
The effects of PKC isoforms on the TGase 1
transcriptional activity. The reporter pd-95 was co-transfected with
the indicated amounts of SRD vector (mock) (A), and
expression vectors for cPKC (B), cPKC
II (C), cPKC
(D), nPKC
(E),
nPKC
(F), and aPKC
(G) into FRSK cells.
After 2 days, luciferase activity was assayed as described under
``Experimental Procedures.'' Data represent the means of
duplicate experiments normalized to
-galactosidase
activity.
Several reports have provided evidence that PKC regulates
TGase 1 gene expression. We found that TPA, an activator of PKC,
increases TGase 1 mRNA in cultured normal human epidermal keratinocytes
and this effect is suppressed by the PKC inhibitors H-7 and
staurosporine (20) . Dlugosz and Yuspa (26) have also
shown that the effect of TPA is blocked by the PKC inhibitors,
GF109203X and bryostatin 1, in cultured primary mouse epidermal
keratinocytes. They also postulated that TGase 1 mRNA expression is
regulated at the transcriptional level, from the results of a nuclear
run-on assay. The transcriptional regulation by TPA has been confirmed
in rabbit and human TGase 1 genes by means of a transient expression
assay using the 5` upstream region of the genes (21, 38) . In this study, we show further direct
evidence that nPKC, an isoform of PKC, regulates the
transcriptional activation of the TGase 1 gene. This mechanism may also
be functional in the epidermis of the skin, because the localization of
nPKC
is closely associated with that of TGase 1 mRNA in normal
and psoriatic
epidermis(16, 17, 34, 43) .
Deletion of the 5` upstream region of the human TGase 1 gene
indicates that the sequence from -95 to -67 includes
critical elements in response to both nPKC and TPA. The
overexpression of nPKC
with TPA induces no additional
potentiation of its transcription in FRSK and nPKC
without the
kinase domain cannot increase the transcriptional activity. Thus, it is
likely that TPA activates nPKC
and the signal generated by
phosphorylation is concentrated on the elements of the upstream region.
Saunders et al.(21) have demonstrated the promoter
activity of the proximal upstream region of rabbit TGase 1 gene. Its
activation requires the sequence from -95 to -50 of the
gene, the position of which is similar to that of human TGase 1 gene (12) . However, the homology of their sequences was not so
high, suggesting a difference in TGase 1 gene regulation between the
species.
As shown by immunoblotting, exogenous nPKC is well
expressed in FRSK as well as HT-1080 fibroblastic cells. An increase in
the expression levels of nPKC
by exogenous introduction of an
nPKC
expression plasmid activates the transcription of TGase 1 in
FRSK cells, but to a very limited extent in HT-1080 cells. Therefore,
the signaling for TGase 1 gene expression is probably mediated by
nPKC
, but its presence is not necessarily sufficient to induce
the transcriptional activation of the gene.
In terms of ability to
induce TGase 1 transcription, nPKC was the most potent of all the
PKC isoforms tested, cPKC
, cPKC
II, cPKC
, nPKC
, and
aPKC
. This prominent efficiency of nPKC
seems to represent
its selective importance in epidermal differentiation. It is localized
in rough endoplasmic reticulum(48) , and, unlike cPKCs, it
cannot be translocated to the plasma membrane by
activation(45) . This unique feature of nPKC
may be
related to the induction of TGase 1 transcription. We also find that
nPKC
can induce TGase 1 transcription to some extent, suggesting
possible contribution of the gene expression, although the PKC isoform
is expressed in various organs including
epidermis(45, 47) .
There is neither a conventional
nor other TPA-responsive elements in the 5` upstream sequence from
-95 to -67, which is critical to TGase 1
transcription(12) . We pointed out two Sp1 motifs in the 5`
proximal upstream region of human TGase 1 gene(12) . Indeed,
when a Sp1 expression plasmid was introduced into FRSK cells with a
reporter plasmid pd-95, the luciferase activity increased (data not
shown). Hence, we speculate that Sp1 or Sp1-related factors are
involved in the transcriptional activation of TGase 1 gene. Further
studies on the characterization of the signaling system via nPKC
will provide new insights in the control of TGase 1 gene expression,
which will lead to the elucidation of the molecular mechanisms involved
in epidermal terminal differentiation and the molecular pathogenesis of
keratinizing disorders, including lamellar ichthyosis.