(Received for publication, July 13, 1994; and in revised form, December 20, 1994)
From the
In this study, we show that all-trans-retinoic acid
(RA) is a potent inducer of tissue transglutaminase (TGase II) and
apoptosis in the rat tracheobronchial epithelial cell line SPOC-1. We
demonstrate that these cells express the retinoid receptors RAR,
RAR
, and RXR
. To identify which of these receptors are
involved in regulating these processes, we analyzed the effects of
several receptor-selective agonists, an antagonist, and a
dominant-negative RAR
. We show that the RAR-selective retinoid
SRI-6751-84 strongly increased TGase II expression at both the
protein and mRNA levels, whereas the RXR-selective retinoid SR11217 had
little effect. The RAR
-selective retinoid Ro40-6055 was also
able to induce TGase II, whereas the RAR
-selective retinoid CD437
was inactive. The induction of TGase II by the RAR-selective retinoid
was completely inhibited by the RAR
-antagonist Ro41-5253.
Overexpression of a truncated RAR
gene with dominant-negative
activity also inhibited the induction of TGase II expression. The
increase in TGase II is associated with an induction of apoptosis as
revealed by DNA fragmentation and the generation of apoptotic cells. We
demonstrate that apoptosis is affected by retinoids in a manner similar
to TGase II. Our results suggest that the induction of TGase II
expression and apoptosis in SPOC-1 cells are mediated through an
RAR
-dependent signaling pathway.
Transglutaminases (EC 2.3.2.13, TGases) ()are
Ca
-dependent enzymes catalyzing the formation of
(
-glutamyl)lysine cross-links between polypeptide
chains(1) . Several members of this gene family have been
identified including the type I (epidermal) TGase (2, 3) and type II (tissue)
TGase(4, 5) . TGase I is induced during squamous
differentiation and plays a role in the formation of the cross-linked
envelope(2, 6) . The function of TGase II is less well
established. TGase II has been implicated in the activation of several
cytokines (7, 8) and in signal
transduction(9) . Expression of TGase II has been found in
association with apoptosis in several cell types, and a role for TGase
II in this process has been
suggested(10, 11, 12) . Apoptosis is a
genetically controlled process of cell death and is important in the
elimination of cells during morphogenesis in embryonic development as
well as in many adult tissues(12, 13) . Although the
function of TGase II in apoptosis has yet to be elucidated, it has been
proposed that TGase II may cross-link cellular proteins, thereby
preventing the release of intracellular macromolecules(14) .
RA has been reported to induce TGase II expression in a variety of
cell
types(4, 15, 16, 17, 18, 19, 20, 21, 22, 23) .
In macrophages, the regulation of TGase II expression by RA occurs at
the transcriptional level(4) . It is believed that many of the
effects of RA on gene expression are mediated by the activation of
nuclear retinoid receptors, RARs and/or RXR's (24) . The
RAR and RXR gene family each comprises three subtypes named ,
, and
(24) . These subtypes are expressed in a
developmental stage- and cell type-specific manner(24) , and
each may regulate the expression of different genes.
In this study,
we examined the retinoid signaling pathways that are involved in the
induction of TGase II and apoptosis in rat tracheobronchial epithelial
SPOC-1 cells utilizing several novel retinoid receptor-selective
agonists and an antagonist. We have also addressed this question
through the expression of a truncated RAR that acts as a
dominant-negative receptor(25) . Our results provide evidence
indicating that the induction of TGase II gene expression and apoptosis
by RA is mediated through a specific retinoid signaling pathway that
involves RAR
.
Figure 1:
Regulation of TGase I
and TGase II by RA in rat tracheal epithelial SPOC-1 cells. A,
Confluent cultures were treated for 4 days with RA at the indicated
concentrations. Then, total cellular protein was isolated and examined
by immunoblot analysis using the TGase I antibody B.C1 or the TGase II
mouse monoclonal antibody Cub 7401 as described under
``Experimental Procedures.'' B, confluent cultures
were treated with 10M RA for the times
indicated and then examined for TGase II by immunoblot
analysis.
Figure 2:
Expression of RARs and RXRs in SPOC-1
cells. Confluent cultures were treated for 4 days with 10M RA, the RAR-selective retinoid SRI-6751-84 (RAR-Sel.) or vehicle (Control). Poly(A)
RNA (5 µg) was fractionated, transferred to a Hybond
membrane, and hybridized to
P-labeled cDNA probes for
mouse RAR
, -
, and -
, human RXR
, -
, and -
,
and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Figure 3:
Transactivation of RARE-tk-LUC and
RXRE-tk-CAT by RA and RAR- and RXR-selective retinoids,
SRI-6751-84 (RAR Sel.) and SRI-11217 (RXR
Sel.). SPOC-1 cells transfected with
RARE-tk-LUC (A)
and RXRE-tk-CAT (B) were treated with dimethyl sulfoxide or
different retinoids at the concentrations indicated. After 48 h of
treatment, LUC or CAT activity was measured as described under
``Experimental Procedures.''
-Actin-CAT or
-actin-LUC was included in all transfections to normalize for
differences in transfection efficiency. Error bars indicate
S.E.
Figure 4:
Induction of TGase II protein by retinoid
receptor-selective ligands in SPOC-1 cells. Confluent cultures were
treated with SRI-6751-84 (RAR sel.), Ro40-6055 (RAR- sel.), CD437 (RAR-
sel.), or
SRI-11217 (RXR sel.) at the indicated concentrations for 4
days, cells were then collected and analyzed for the presence of TGase
II protein by immunoblot analysis.
The increase in TGase II protein
levels by retinoids was related to increased levels of the
corresponding mRNA. Northern blot analysis revealed that treatment with
the RAR-selective retinoid SRI-6751-84 increased the level of
TGase II mRNA in a dose-dependent manner (Fig. 5). The EC for this stimulation was about 1-2
10
M, which is similar to that for the effect of
SRI-6751-84 on TGase II protein levels (compare row 1 in Fig. 4with Fig. 5). However, treatment of SPOC-1 cells
with the RXR-selective retinoid SR11217 failed to enhance TGase II mRNA (Fig. 5). These results are consistent with the observations
obtained from the immunoblot analysis (compare row 4 in Fig. 4with Fig. 5).
Figure 5: Dose-dependent induction of TGase II mRNA expression by receptor-selective retinoids in SPOC-1 cells. Confluent cells were treated with the RAR-selective retinoid SRI-6751-84 and the RXR-selective retinoid SRI-11217 at the concentrations indicated. After 4 days of treatment, total RNA was isolated, fractionated on 1.0% agarose gel (30 µg/lane), and then transferred to a Hybond membrane. The membrane was successively probed with cDNAs for TGase II and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
To examine whether the
RXR-selective retinoid influences the induction of TGase II by the
RAR-selective retinoid, confluent cultures were treated with a
suboptimal concentration of SRI-6751-84 (10M) in the presence of increasing concentrations of the
RXR-selective retinoid SR11217 for 4 days and then analyzed for TGase
II protein. Immunoblot analysis revealed that the RXR-selective
retinoid had no effect on the induction of TGase II expression by the
RAR-selective retinoid (data not shown).
Figure 6:
Effect of the RAR-selective
antagonist Ro41-5253 on the transactivation of
RARE-tk-LUC
and the regulation of TGase II. A, SPOC-1 cells transfected
with
RARE-tk-LUC were treated with a 10
M concentration of the RAR-selective retinoid SRI-6751-84 (RAR Sel.) and 2
10
M Ro41-5253 (Antag.) in the combinations indicated.
After 48 h of treatment, cells were collected and the relative LUC
activity was determined. B, confluent cells were treated with
the RAR-selective ligand (RAR Sel.) in the presence of
increasing concentrations of Ro41-5253 (RAR-
antag.). After 4 days of treatment, total cellular protein was
isolated and examined for TGase II expression by immunoblot
analysis.
Figure 7:
Suppression of the retinoid-induced
transactivation and TGase II expression in SPOC-1 cells by
overexpression of the truncated RAR gene RAR
403. A,
RAR
403 expression in LRAR
403SN-infected SPOC-1 cells.
Poly(A)
RNAs from parental SPOC-1 cells and LXSN- and
LRAR
403SN-infected cells were examined by Northern blot analysis
using a radiolabeled cDNA probe for RAR
. B, effect of
SRI-6751-84 (RAR-sel.) and Ro40-6055 (RAR
-sel.) on the transactivation of
RARE-tk-LUC in
parental and LXSN- and LRAR
403SN-infected SPOC-1 cells.
Transfected cells were treated for 24 h with retinoids and then assayed
for LUC activity. Results shown are representative for three
independent experiments. C, comparison of TGase II induction
in parental and LXSN- and LRAR
403SN-infected SPOC-1 cells. Cells
were treated with SRI-6751-84 at indicated concentrations for 4
days and examined for TGase II expression by immunoblot analysis. In
this experiment, the induction of TGase II at 10
M SRI-6751-84 is higher than that seen in the
experiment shown in Fig. 4due to experimental
variation.
Next we analyzed the
effect of RAR403 on the
RARE-dependent transactivation of CAT (Fig. 7B) and TGase II induction (Fig. 7C) by retinoids. In the parental and
LXSN-infected (control) SPOC-1 cells, the RAR-selective
(10
M) and RAR
-selective
(10
M) retinoids induced CAT activity to
about the same extent. In the LRAR
403SN-infected cells, the
transactivation by the RAR-selective retinoid was about 50% reduced
whereas the transactivation by the RAR
-selective retinoid was
completely abolished. The partial inhibition of the transactivation
induced by the RAR-selective retinoid might be due to the fact that the
transactivation mediated through RAR
is not or is only partially
inhibited. As shown in Fig. 7C, SRI-6751-84
induced TGase II equally well in LXSN-infected and parental SPOC-1
cells. However, the induction of TGase II in LRAR
403SN-infected
SPOC-1 cells was considerably less sensitive to SRI-6751-84 than
that in LXSN-infected cells (Fig. 7C). In
LRAR
403SN-infected cells, 10
M SRI-6751-84 was necessary to induce TGase II. This
concentration is about 100-fold higher than that required for the
LXSN-infected or the parental SPOC-1 cells.
Figure 8:
Electrophoretic analysis of DNA
fragmentation in SPOC-1 and human lung carcinoma cells. SPOC-1 cells
were treated with 10M RA, 10
M RAR- or RXR-selective retinoids or 2
10
M RAR
antagonist in the presence or
absence of the RAR-selective retinoid for 4 days, and then DNA was
isolated and separated by 1% agarose gel electrophoresis. A,
parental SPOC-1 cells; B, LRAR
403SN-infected SPOC-1
cells; C, human lung carcinoma NIH-HUT 460 cells. Cells were
treated with RA (10
M) or TGF
1 (100
pM) for 3 days.
Previously, we
showed that RA and TGF1 induce TGase II in several human lung
carcinoma cell lines, including adenocarcinoma NCI-H460
cells(22) . Therefore, we wished to investigate whether the
induction of TGase II by RA is also accompanied by apoptosis in these
cells. As shown in Fig. 8C, treatment of NCI-H460 cells
with RA or TGF
1 resulted in the formation of fragmented DNA. These
findings indicate that the induction of apoptosis by RA is not
restricted to SPOC-1 cells and strengthen the correlation between the
induction of TGase II and apoptosis.
To obtain further support for
the induction of apoptosis by retinoids in SPOC-1 cells, the
retinoid-treated and untreated SPOC-1 cells were examined for the
presence of apoptotic cells by electron microscopy. Few apoptotic cells
(<0.1%) were observed in control cultures and in cultures treated
simultaneously with the RAR antagonist and the RAR-selective
ligand or treated with the RXR-selective retinoid. However, an
increased number of apoptotic cells (3-5%) was observed in
cultures treated with RA or the RAR-selective retinoid
SRI-6751-84. Fig. 9B shows the typical morphology of
an apoptotic cell at an early stage of apoptosis in cultures treated
with SRI-6751-84. In contrast to the control cell (Fig. 9A), most of the chromatin in the apoptotic cell is
aggregated in large compact and segregated granular masses that abut on
the nuclear membrane. The cytoplasm is much more condensed than that of
the control cell, resulting in crowding of the organelles, which retain
their integrity.
Figure 9:
Electron micrograph of a normal SPOC-1
cell (A) (magnification 9,000) and an apoptotic cell (B)(magnification
11,200) at an early stage of
apoptosis in cultures treated with 10
M
RAR-selective retinoid for 4 days. Note the condensation and
segregation of nuclear chromatin into compact masses abut the nuclear
membrane, some of which start to protrude from the
membrane.
RA has been reported to induce TGase II gene expression in
many cell types including several tracheobronchial epithelial cell
lines (15, 16, 17, 18, 19, 20, 21, 22) .
Although the molecular basis of this regulation has not yet been
elucidated, it is likely that retinoid receptors are implicated in this
action. In the present study, we examined the signaling pathways that
are involved in the retinoid-induced increase of TGase II and apoptosis
in rat tracheal epithelial SPOC-1 cells. We demonstrate that these
cells express the nuclear retinoid receptors, RAR, RAR
, and
RXR
, and, after retinoid treatment, also RXR
(Fig. 2).
The effects of several receptor-selective retinoids, an RAR
antagonist and a dominant-negative, truncated RAR
gene were
studied in order to identify the receptor(s) that are involved in these
actions. Our studies indicate that the induction of TGase II gene
expression in SPOC-1 cells by retinoids is mediated by a specific
retinoid signaling pathway that involves the RAR
receptor. Several
lines of evidence support this conclusion. First, the RAR-selective
retinoid is a very potent inducer of TGase II (Fig. 4). This
retinoid binds specifically to RAR but not to RXR receptors (27) and activates
RARE-tk-LUC but not RXRE-tk-CAT (Fig. 3)(28) . In contrast, the RXR-selective retinoid
which selectively binds to RXRs and induces formation of RXR homodimers (28) was unable to induce TGase II (Fig. 4). However,
this retinoid exhibits activity in SPOC-1 cells as indicated by its
ability to transactivate RXRE-tk-CAT (Fig. 3B). These
observations suggest that activation of RARs plays a role in the
induction of TGase II expression by retinoids; however, they do not
rule out the involvement of RXR
or
since RARs mediate their
activity through a complex with RXRs (46) or other nuclear
transcriptional factors(47, 48) . These results
suggest that activation of RARs rather than RXRs is a requirement for
the induction of TGase II by retinoids in tracheobronchial epithelial
cells. This conclusion is in agreement with previous studies showing
increased induction of TGase II in rat tracheal 2C5 cells (20) and 3T3 fibroblasts (23) transfected with RAR
expression vectors. However, the induction of TGase II in the
hematopoietic cell line HL60 cells appears to be dependent on an
RXR-dependent pathway since TGase II is induced by RXR-selective
retinoids but not by RAR-selective retinoids. (
)
Second,
the induction of TGase II by the RAR-selective retinoid was completely
antagonized by the RAR antagonist (Fig. 5). The precise
mechanism by which the antagonist exerts its effect has not yet been
elucidated; however, the antagonist has been shown to compete with RA
specifically for binding to RAR
(30) . Gel-shift
experiments indicate that in the presence of the RAR
-antagonist
the RXR
RAR complex retains its ability to bind to the
RARE(30) . However, the antagonist may be unable to induce the
right conformational change in the RAR
that allows
transactivation(30) . The inhibition of the
SRI-6751-84-induced transactivation of
RARE-tk-LUC by this
antagonist (Fig. 6A) is in agreement with this concept.
The partial inhibition may be due to the fact that the activation
mediated by RAR
is not inhibited by the antagonist. This is
supported by a previous report showing little effect of Ro41-5253
on the
RARE-dependent transactivation by RAR
(30) .
Third, overexpression of the truncated RAR gene RAR
403 in
SPOC-1 cells inhibited
RARE-dependent transactivation and blocked
the induction of TGase II by the RAR-selective retinoid (Fig. 7C). The truncated receptor reduced the
transactivation of
RARE-tk-LUC by the RAR-selective retinoid by
only 50% but completely suppressed the transactivation by the
RAR
-selective retinoid (Fig. 7B). These results
suggests that TGase II induction occurs through RAR
. Previously,
it was reported that the truncated RAR
403 gene blocks the
RA-induced neutrophilic differentiation in HL-60 cells (25) and
acts as a dominant-negative receptor. The mechanism by which the
truncated RAR
exerts its dominant-negative action has yet to be
elucidated. RAR
403 is still able to bind RA in in vitro binding assays (49) although with a 12-fold lower binding
affinity as the normal RAR
(49) . It has been demonstrated
that the RARs mediate their action as part of a heterodimeric complex
with RXR or other transcriptional factors (46, 47, 48) . The product of the
LRAR
403 gene that contains a truncation at its carboxyl terminus
is still able to form heterodimers with the RXR receptor(50) .
Overexpression of the RAR
403 may compete for RXR binding with
endogenous RARs and inhibit the formation of RAR
RXR heterodimers
and, therefore, the activation of gene transcription. However, it
should be noted that RAR
403 caused only a 50% reduction of the
RARE-dependent transactivation by the RAR-selective retinoid and
did not block the effect of RA on several other genes in SPOC-1 cells.
We have found that the effects of retinoids on certain squamous
cell-specific genes are not influenced by RAR
403(43) .
These results indicate that the inhibition caused by the RAR
403 is
specific. This specificity may depend on the retinoid signaling pathway
that controls the target gene or the type of promoter involved.
Fourth, the RAR-selective retinoid Ro40-6055 strongly
induced TGase II expression (Fig. 4). This retinoid was about
100 times less active than SRI-6751-84, which is probably related
to the reported lower affinity of this RAR
-selective retinoid to
RAR
(29) . Although the RAR
-selective retinoid CD437
was able to transactivate
RARE-tk-LUC in SPOC-1 cells as
effectively as Ro40-6055, it was unable to induce TGase II up to
concentrations of 10
M. The differential
responsiveness of TGase II and
RARE-tk-LUC to CD437 may be due to
differences between the sequence or context of the response elements
that determine activation by RAR
. Since expression of RAR
was
neither detectable nor inducible by RA in SPOC-1 cells, our results
indicate that an RAR
-dependent rather than an RAR
- or
RAR
-dependent signaling pathway is involved in the induction of
TGase II expression by RA in SPOC-1 cells.
Although the functions of
TGase II have yet to be fully established, recent studies have
implicated TGase II in the activation of interleukins (7) and
TGF1 (8) and in signal transduction(9) . In
several cell types, the expression of TGase II is associated with the
induction of
apoptosis(10, 11, 12, 14, 51, 52, 53) .
However, this correlation is not absolute. Our study demonstrates for
the first time that retinoids induce apoptosis in rat and human
tracheobronchial epithelial cells as shown by the induction of DNA
fragmentation (DNA ladder) (Fig. 8) and by the appearance of
cells with typical apoptotic features (Fig. 9). In both SPOC-1
and NCI-H460 cells, this induction is accompanied by an increase in
TGase II expression (Fig. 1, Fig. 4, and Fig. 5and (22) ). The pattern by which retinoids induce
apoptosis appears to be identical with that for TGase II. Apoptosis was
induced by the RAR-selective retinoid and inhibited by the RAR
antagonist and by the overexpression of the truncated RAR
gene in
a manner similar to TGase II. Future studies have to determine what the
role is of TGase II in these cells.
Our results demonstrate that
SPOC-1 cells are able to undergo two different processes of
irreversible growth arrest, namely squamous differentiation and
apoptosis. A schematic representation of the differential regulation of
squamous differentiation and apoptosis, and TGase I and TGase II by RA
is shown in Fig. 10. RA inhibits squamous cell differentiation
as shown by the inhibition of TGase I expression, a squamous
cell-specific gene encoding an enzyme that catalyzes the formation of
the cross-linked envelope(2, 6) . At the same time, RA
is able to induce TGase II and initiate an alternative pathway of cell
death, apoptosis (Fig. 1, 4, 5, 8, and 9). These two processes
appear to be regulated by different retinoid signaling pathways. This
is indicated by the difference in the responsiveness of several
squamous-specific genes, such as TGase I (Fig. 1), to the
receptor-selective retinoids compared to that of TGase II and
apoptosis. In contrast to TGase II and apoptosis, the expression of
TGase I and cornifin is regulated by both the RAR- and RXR-selective
retinoids and not affected by the truncated RAR. (
)
Figure 10: Schematic representation of the differential regulation of squamous differentiation and apoptosis and TGase I and TGase II by RA in epithelial cells.
In squamous differentiating tissues, a balance exists between the rate of proliferation and differentiation. It has been suggested that retinoids may control the rate at which cells undergo squamous cell differentiation(54) . The regulation of apoptosis may be another mechanism by which retinoids control homeostasis in these tissues.