* Laboratory of Molecular Embryology, National Institute of Child Health and Human Development, Bethesda, Maryland
20892-5431; and Department of Immunology, Holland Laboratory, American Red Cross, Rockville, Maryland 20855
Thyroid hormone (T3 or 3,5,3-triiodothyronine) plays a causative role during amphibian metamorphosis. To investigate how T3 induces some cells to
die and others to proliferate and differentiate during
this process, we have chosen the model system of intestinal remodeling, which involves apoptotic degeneration of larval epithelial cells and proliferation and differentiation of other cells, such as the fibroblasts and
adult epithelial cells, to form the adult intestine. We
have established in vitro culture conditions for intestinal epithelial cells and fibroblasts. With this system, we
show that T3 can enhance the proliferation of both cell
types. However, T3 also concurrently induces larval epithelial apoptosis, which can be inhibited by the extracellular matrix (ECM). Our studies with known inhibitors of mammalian cell death reveal both similarities
and differences between amphibian and mammalian
cell death. These, together with gene expression analysis, reveal that T3 appears to simultaneously induce different pathways that lead to specific gene regulation,
proliferation, and apoptotic degeneration of the epithelial cells. Thus, our data provide an important molecular and cellular basis for the differential responses of
different cell types to the endogenous T3 during metamorphosis and support a role of ECM during frog
metamorphosis.
ORGANOGENESIS and tissue remodeling require not
only extensive cell proliferation and differentiation, but also selective elimination of unwanted
cells. Such cell removal occurs through well-controled genetic programs, leading to programmed cell death (apoptosis) with a series of distinguished morphological changes (Wyllie et al., 1980 Amphibian metamorphosis is one of the best studied developmental systems where extensive cell removal occurs
(Dodd and Dodd, 1976 Thyroid hormone (T3 or 3, 5, 3 To investigate how T3 controls cell fate during tissue remodeling, we have established conditions for in vitro cultures of tadpole intestinal epithelial and fibroblastic cells.
Addition of T3 to the culture medium causes cell death of
the larval epithelial cells with typical apoptotic morphology. In contrast, fibroblastic cells are refractory to the hormone-induced cell death; instead, T3 induces the proliferation of those cells. We further show that the epithelial
apotosis in vitro can be blocked by some known inhibitor
of mammalial cell death and by ECM.
Isolation and Culturing of Tadpole Intestinal Epithelial
and Fibroblastic Cells
Tadpole intestinal fragments were isolated from the posterior small intestine of premetamorphic tadpoles at stage 57/58 (Nieuwkoop and Faber,
1956 To isolate both epithelial cells and fibroblasts, the anterior small intestinal fragments were digested and cultured as above. The anterior small
intestine contains the single intestinal fold where connective tissue is
abundant (Marshall and Dixon, 1978 It should be pointed out that the epithelial cells isolated from the anterior and posterior small intestine behaved identically in vitro in the presence or absence of T3 (see Results), and were thus used without distinction in this study.
DNA Fragmentation Assessment by ELISA
The tadpole intestinal epithelial cells or fibroblasts isolated above were labeled overnight in the presence of 10 µM of 5-bromo-2 DNA Content Analysis
The primary culture of tadpole intestine epithelial cells was treated with
or without 100 nM T3. The cells were harvested by trypsinization and fixed
in 1% formaldehyde in PBS on ice for 15 min. After centrifugation, the
cell pellets (~5 × 105 cells) were suspended in 2 ml of 0.1% sodium citrate-0.3% NP-40 solution, pH 7.5, and were passed through a 60-µM nylon mesh (Spectramesh; Fisher Scientific Co., Pittsburgh, PA). The cells
were stained with 50 µg/ml of propidium iodide and then treated with 50 µg/ml of RNase A at 37°C for 30 min. The fluorescence intensity of individual cells was measured with a flow cytometer (FacScan Immunosystem; Becton Dickinson, Franklin Lakes, NJ).
Agarose Gel Electrophoresis Analysis of DNA
Fragmentation during Cell Death
The primary epithelial cells were cultured with or without 100 nM T3 for 1 d.
The cells were collected by centrifugation at 500 g for 5 min at 4°C and
then lysed in 10 mM Tris-HCl, pH 8, 100 mM NaCl, 25 mM EDTA, 0.5%
sodium dodecyl sulfate, and 0.1 µg/ml proteinase K. The lysate was incubated overnight at 50°C. After extraction with an equal volume of phenol/
chloroform/isoamyl alcohol (25:24:1), the DNA in the lysate was precipitated with ethanol, redissolved in H2O, and treated with RNase A (DNase
free, 10 µg/ml) at 37°C for 2 h. The sample was again extracted with an
equal volume of phenol/chloroform/isoamyl alcohol and precipitated with
ethanol. 20 µg of the final purified DNA were fractionated on a 1.2% agarose
gel, stained with ethidium bromide, and visualized under ultraviolet light.
Cell Proliferation Assay
Intestinal epithelial cells or fibroblasts were cultured overnight at 25°C in
96-well plastic plates or 6-well plates with or without different matrix coating (5 × 104 cells/well) in the presence of or absence of 100 nM T3 and/or 600 ng/ml CsA. [3H]Thymidine was added at 1 µCi/ml. After another 5 h
at 25°C, the cells were then lysed by repeated freezing and thawing. The [3H]thymidine incorporated into genomic DNA was then measured by scintillation counting.
Cell Culturing on Matrix-coated Plastic Dishes
The epithelial cells were cultured on 6-well plastic plates coated with various matrices (Becton Dickinson Labware, Bedford, MA; 1-50 × 104 cells/
well) in the presence or absence of indicated concentrations of T3. For cell
death measurement, the cultured cells were isolated by trypsinization and
then pelleted and lysed. The lysates were transferred to a 96-well dish for
the ELISA assay. For cell proliferation assay, 10 µCi [3H]thymidine was
added after overnight treatment with or without T3 and then the cells were
incubated in the 1-ml medium for another 5 h. The cells were then isolated
by trypsinization and then pelleted and lysed for [3H]thymidine incorporation assay.
RNA Isolation and Analysis
Intestinal epithelial cells from stage 57/58 or stage 64 tadpoles were cultured on plastic dishes with or without ECM coatings in the presence or
absence 100 nM T3 and/or 600 ng/ml CsA or 10 ng/ml FK506. After 1 d of
culturing, the total RNA was isolated by using RNAzol (Tel-Test, Inc.,
Friendwood, TX) and quantified by absorption at 260 nM.
Total RNA was electrophoresed on a 1% agarose-formaldehyde gel
and transferred onto a GeneScreen membrane (NEN Life Science Products, Boston, MA) after partial hydrolysis with NaOH (Maniatis et al., 1982 Cell Type-specific Responses to Thyroid Hormone in
Primary Intestinal Cell Cultures
To investigate how T3 induces the degeneration of larval
epithelium and proliferation and differentiation of adult
cell types in the intestine, we dissociated the anterior small
intestine of stage 57/58 Xenopus laevis tadpoles and isolated both the epithelial cells and the rest of the intestinal
cells, which were predominantly mature and immature fibroblasts (McAvoy and Dixon, 1977
A major property of programmed cell death in mammals is the formation of a ladder of multinucleosomal-sized genomic DNA fragments. To determine whether T3-induced larval epithelial cell degeneration in vitro also
possesses such changes, genomic DNA was isolated from
epithelial cells cultured for 1 d in the presence or absence of T3 and analyzed on an agarose gel. The results clearly
showed that T3 induced a nucleosomal DNA fragmentation ladder (Fig. 2 A).
Using an ELISA assay designed to measure the extent
of nuclear DNA fragmentation, we found that T3 caused
epithelial cell death in a dose-dependent manner, with extensive cell death occurring at physiological concentrations (5-10 nM; Leloup and Buscaglia, 1977 T3 Stimulates the Proliferation of Both Larval
Epithelial Cells and Fibroblasts
Although differentiated and fully functional, larval intestinal epithelial cells are capable of proliferation (McAvoy
and Dixon, 1977 To directly investigate the possible effect of T3 on intestinal cell proliferation, the [3H]thymidine incorporation assay was performed. Both the epithelial cells and fibroblasts
had similar levels of [3H]thymidine incorporated in the absence of T3 (Fig. 4). T3 treatment stimulated [3H]thymidine incorporation in both the epithelial cells and fibroblasts to a similar extent. Thus, both epithelial cells and
fibroblasts can proliferate in vitro with similar rates, and
T3 causes nearly a twofold increase in this proliferation for
these two cell types of the tadpole intestine.
Gene Regulation by T3 in In Vitro Epithelial
Cell Culture
T3 is known to regulate gene expression during amphibian
metamorphosis (Shi, 1994 When the intestinal epithelial cells from stage 57/58 tadpoles were cultured in the presence of 100 nM T3, the levels of IFABP mRNA were not significantly affected within
16 h, but downregulated after 24 h (Fig. 5 A), in agreement
with the suggestion that IFABP gene is not a direct T3-
response gene (Shi and Hayes, 1994
The surprising result was, however, that the expression
of both the IFABP and Na+/PO43
ECM Inhibits Epithelial Cell Death In Vitro
The intestinal epithelium is in close contact with the basal
lamina, a special ECM that separates the epithelium from
the underlying mesenchyme. It is known that the basal
lamina undergoes extensive remodeling during metamorphosis (Ishizuya-Oka and Shimozawa, 1987b
Similar to many other types of cells, the fate of the tadpole intestinal epithelial cells is likely to be controled by a
balance of survival and death factors. The enhanced survival of the intestinal epithelial cells on matrix-coated dishes
implies that these matrices may inhibit T3-induced epithelial apoptosis. To test this possibility, tadpole intestinal
epithelial cells were cultured on fibronectin- (Fig. 7 A) or
type I collagen- (data not shown) coated dishes and treated
with T3. Although T3 treatment still led to epithelial cell
death, >50 and 10% of the cells remained viable after 2 and 4 d of treatment, respectively (Fig. 7 B). Under the
same conditions, all of the cells cultured on plastic dishes
died after 3 d of treatment (Fig. 1 B). Quantitative analysis
of the T3-induced apoptosis by ELISA assay revealed that
all matrices inhibited ~50% of the T3-induced DNA fragmentation after 2 or 3 d of T3 treatment (Fig. 8 A). On
the other hand, the T3-induced epithelial apoptosis on different coatings had similar T3-dose responses (Fig. 8 B),
indicating the effects of these matrices were not simply
due to any possible effects of the matrices on the availability of T3. Instead, the results suggest that the matrices were
influencing cellular responses to T3 through yet unknown
signal transduction pathways.
In contrast to the effects of ECM on T3-induced cell death,
3H-thymidine incorporation showed that the various matrices had little effect on T3-induced cell proliferation (Fig.
8 C). Similarly, they had no effect on the downregulation
of IFABP gene expression by T3 (Fig. 8 D). These results
suggest that T3 induces multiple cellular events, each of
which is in turn affected by different signal transduction
pathways.
Cell Death Inhibition Studies Confirm the Existence of
Multiple Signal Transduction Pathways Induced by T3
The results above suggest that the differential responses to
T3 of different intestinal cells during amphibian metamorphosis lie mainly with the ability of T3 to induce epithelial
apoptosis. The exact mechanism underlying apoptosis remains unknown despite extensive investigations. However,
earlier studies in mammals have demonstrated the involvement of ICE-like proteases and nucleases during
programmed cell death (Martin and Green, 1995 Both Z-VAD and ATA inhibited the T3-induced intestinal epithelial cell death (Figs. 9, A and B), suggesting the
participation of ICE-like proteases and nucleases, respectively. Of the two immunosuppressants, only CsA inhibited the T3-induced epithelial apoptosis (Figs. 9, A and B).
Identical results were obtained with different concentrations of these drugs (data not shown). Thus, CsA has similar
effects on T3-induced apoptosis as on activation-induced T cell death, whereas FK506 has different effects on these
two apoptotic processes.
Interestingly, using flow cytometry we observed that
CsA blocked the apoptosis of cells at all different stages of
the cell cycle, resulting in a profile of cell distribution similar to that of the control cells in the absence of T3 (Fig. 3 A
and data not shown). In contrast, CsA had no effect on
DNA synthesis both in the presence or absence of T3 (Fig.
9 C). These results suggest that T3 simultaneously induces
the cell death and proliferation in the epithelial cells, and
only the death pathway is sensitive to CsA. Such a conclusion is also consistent with the fact that T3 stimulates fibroblastic cell proliferation even though it does not cause fibroblastic cell apoptosis.
The induction of epithelial apoptosis by T3 is presumably through the activation and/or repression of certain
genes in the intestine. Currently, it is not known which, if
any, of the known T3-regulated genes are involved in epithelial cell death. The ability of T3 to regulate epithelial gene
expression in vitro prompted us to investigate whether the
cell death inhibitors used above can affect T3-dependent
gene regulation. Of particular interest is the immunosuppressants FK506 and CsA. Both drugs are known to inhibit activation-induced T cell death (Shi et al., 1989 We have successfully cultured cells of the tadpole intestine
in vitro and investigated the effects of thyroid hormone on
these primary cell cultures. We have demonstrated here
that both larval epithelial and fibroblastic cells respond to
T3 by increasing their DNA synthesis, and that only the epithelial cells undergo T3-dependent programmed cell death
with typical apoptotic properties as observed in mammals.
This T3-induced epithelial apoptosis can be inhibited by
ECMs. More importantly, our study reveals that T3 induces multiple pathways in the larval epithelial cells.
Primary Cell Cultures of Tadpole Intestine Mimic the
Cell-Specific Responses to T3 in Intact Tadpoles
Organ culture experiments have shown that the regulation
of amphibian metamorphosis by T3 is organ autonomous
(Dodd and Dodd, 1977; Ishizuya-Oka and Shimozawa,
1991 The fibroblasts, on the other hand, are refractory to T3-induced apoptosis in our in vitro system. This agrees well
with their ability to proliferate and differentiate but not
undergo apoptosis during natural metamorphosis (Ishizuya-Oka and Shimozawa, 1987a A surprising finding is that T3 also stimulates the proliferation of the epithelial cells. However, larval intestinal
epithelia cells are known to be capable of dividing in spite
of their differentiated phenotype (McAvoy and Dixon, 1977 Concurrent Induction of Multiple Pathways by T3 in the
Larval Intestinal Epithelial Cells
Amphibian metamorphosis is perhaps one of the processes where cell death takes place at its extreme. All the
tadpole-specific organs, such as the tail and gill, degenerate completely whereas the rest of the organs undergo extensive remodeling or de novo development. Most, if not
all, of the organ transformations require the removal of
some or all of the existing cells. Early microscopic examinations have shown that the tail resorption and intestinal remodeling involve cell death with typical apoptotic morphologies as observed in mammals (Kerr et al., 1974 The induction of intestinal apoptosis by T3 is believed to
be through the activation of the cell death pathway and/or
the deactivation of cell survival signals. Although many thyroid hormone response genes have been identified in the
intestine (Shi and Ishizuya-Oka, 1996 Our studies with immunosuppressants CsA and FK506
show that CsA inhibits T3-induced cell death, similar to
that observed for activation-induced T cell death in mammals (Shi et al., 1989 An intriguing possibility has been suggested by the recent finding that CsA inhibits the DNA binding activity of
the transcription factor Nur77 in T cells (Yazdanbakhsh
et al., 1995 Independent of the exact mechanism of CsA action, the
fact that CsA can block T3-induced cell death while having
no effect on T3-induced downregulation of the IFABP gene,
and an increase in cell proliferation supports the idea that
T3 induces multiple, indepencent cellular events in the intestinal epithelial cells. These include apoptosis, cell proliferation, and specific regulation of genes that are involved in neither cell death nor cell proliferation. Such a
conclusion is also supported by the ability of ECM to inhibit intestinal epithelial cell death but not cell proliferation.
Role of ECM in Epithelial Development during
Intestinal Remodeling
The intestinal epithelium is separated from the mesenchyme
by a special ECM, the basal lamina, whose major components include laminin, entactin, type IV collagen, and fibronectin, etc. (Hay, 1991 Studies in both mammals and amphibians have implicated a role of basal lamina during intestinal development
(for review see Louvard et al., 1992 Our results indicate that basal lamina components laminin, fibronectin, and type IV collagen can directly inhibit
T3-induced larval epithelial cell death. Although similar
effects observed with type I collagen were essentially absent in the basal lamina, this may reflect the fact that our
primary epithelial cell cultures represent an extreme case
where all cell-ECM interactions had been removed upon
dissociating the epithelial cells. It is very likely that disrupting the interactions between the epithelial cells and
different ECM components may have different effects on
epithelial behavior in vivo. In this regard, it is interesting to note that a number of matrix metalloproteinase genes
are upregulated during intestinal remodeling and tail resorption (Patterton et al., 1995 Further evidence on the role of ECM in epithelial development comes from our analysis on T3-dependent gene
regulation. During normal development, the intestinal IFABP and Na+/PO43 It is unclear how ECM influences intestinal epithelial
development during metamorphosis. Studies in various
model systems have provided evidence for the involvement of cell surface ECM receptors, especially integrins,
in transducing the ECM signals (Werb et al., 1989; Jacobson et al., 1997
). Extensive studies in recent years have identified and characterized many
of the genes that participate in cell death during various
physiological and pathological processes. However, relatively little is known about how cell death is controlled
spatially and temporally during development, and how cell
specificity of apoptosis is achieved.
; Gilbert and Frieden, 1981
; Gilbert
et al., 1996
). This process systematically transforms different tadpole organs to adult forms. Some tissues such as the
tail are tadpole specific and are completely resorbed during metamorphosis. Others, like the hindlimb, develop de
novo from undifferentiated blastema cells. The rest of the organs, such as the intestine, are present in both the
premetamorphic tadpoles and post metamorphic frogs, but
are drastically remodeled during metamorphosis (Dodd and
Dodd, 1976
; Dauca and Hourdry, 1985
; Yoshizato, 1989
;
Shi and Ishuzuya-Oka, 1996). Interestingly, cell death appears to take place in all three types of transformations, although most dramatically during organ resorption. Early
studies, particularly microscopic examinations, have revealed that cell death during tissue resorption and remodeling occurs through apoptosis (Kerr et al., 1974
; Ishizuya-Oka and Shimozawa, 1992a
; Ishizuya-Oka and Ueda,
1996
; Izutsu et al., 1996
). However, the lack of a proper in
vitro system has so far hampered the understanding of the
molecular mechanism underlying this apoptotic process.
-triiodothyronine)1 plays
an essential causative role during amphibian metamorphosis (Gilbert and Frieden, 1981
; Kikuyama et al., 1993
; Gilbert et al., 1996
). The hormone is known to directly regulate gene transcription through thyroid hormone receptors
(TRs), which are nuclear transcription factors (Tsai and
O'Malley, 1994
; Yen and Chin, 1994
; Mongelsdorf et al.,
1995; Shi et al., 1996
). Many of the genes that are regulated by T3 during metamorphosis have been identified and characterized (Shi, 1994
; Brown et al., 1996
; Gilbert et al., 1996
).
Among the so-called early response genes (those that change
their mRNA levels within 1 d of T3 treatment of premetamorphic tadpoles) there are genes encoding transcription
factors, extracellular matrix (ECM) modification/digestion
enzymes, and ECM components (Shi, 1994
; Brown et al.,
1996
; Gilbert et al., 1996
). Noticeably absent among them
are genes directly involved in programmed cell death, such as interleukin-1
-converting enzyme (ICE)-like proteases
and Bcl-2 familiar members, etc. (White, 1996
). Although
it is possible that such genes are among the yet to be identified early T3 response genes, it is more likely that such
genes are further downstream.
Materials and Methods
) and then digested with collagenase and dispase (Ishizuya-Oka and
Shimozawa, 1992b
). The dissociated cells (predominantly epithelial cells,
>80%) were cultured overnight at 25°C on plastic dishes in 60% L15 medium supplemented with 10% fetal calf serum (GIBCO BRL, Gaithersburg, MD). The serum was treated with resin (AGI-X8; Bio-Rad Laboratories, Hercules, CA) to remove thyroid hormone (Samuels et al., 1979
).
After overnight culturing, the epithelial cells were then transferred to a
new dish after gentle shaking (tightly attached mesenchymal cells were left
behind).
; Ishizuya-Oka and Shimozawa, 1987a
).
Thus, upon transferring the epithelial cells after overnight culturing on
plastic dishes, the vast majority (>80%) of cells remaining attached to the
dish were fibroblasts (referred to as fibroblasts throughout this article).
-deoxyl-uridine (BrdU) at 25°C. The cells were collected by centrifugation at 250 g and 2 × 104 cells/well were cultured in a 96-well plastic culture plate containing different concentrations of T3 for indicated times. The cells were lysed and
the supernatant was assayed for DNA fragmentation (cellular DNA fragmentation ELISA Kit; Boehringer Mannheim, Mannheim, Germany) according to the manufacturer's instructions.
;
Ranjan et al., 1994
). Hybridization was done by using the cDNA probes of
Xenopus intestinal fatty acid binding protein (IFABP; Shi and Hayes,
1994
), Na+/PO43
cotransporter (Ishizuya-Oka et al., 1997
), and rpL8 (Shi
and Liang, 1994
). After overnight hybridization at 42°C in 50% formamide, 5 × SSPE, 0.2% SDS, 10% dextran sulfate, 5× Denhardt's solution, and 100 µg/ml denatured salmon sperm DNA, the filters were washed three times for 5-10 min each at room temperature in 2 × SSC and 0.2%
SDS. Stringent washes were then done twice for 25 min each in 0.25 × SSC and 0.2% SDS at 65°C.
Results
; Ishizuya-Oka and
Shimozawa, 1987a
, b
). Upon culturing in vitro in the presence of 10% T3-depleted calf serum, the fibroblastic cells
slowly proliferated, with a doubling time of ~2.5 d (Fig.
1 A). In contrast, the viable epithelial cells gradually decreased in number with ~60% of live cells remaining after
4 d of culturing (Fig. 1 B). Interestingly, addition of 100 nM
T3 had contrasting effects on the cultured primary cells.
The T3 treatment doubled the proliferation rate of the fibroblasts (Fig. 1 A) while drastically stimulating the degeneration of the epithelial cells (Fig. 1 B). Even at 10 nM, close to the endogenous plasma concentration at the climax
of metamorphosis (Leloup and Buscaglia, 1977
), T3 caused
considerable reductions in epithelial cell survival (Fig. 1 B).
Fig. 1.
Contrasting effects of thyroid hormone
on tadpole intestinal epithelial and fibroblastic
cells. The fibroblasts (A) and epithelial cells (B)
were isolated from stage 57/58 of tadpole small
intestine and then cultured on a six-well plastic
dish in 60% L-15 medium containing 10% T3-
depleted fetal bovine serum at 25°C in the presence or absence of 10 or 100 nM T3. The live cells
were counted daily by trypan blue staining. Note
that the epithelial cell number decreased even
when no exogenous T3 was present. This could
be because of the residual T3 in the treated serum. DNA fragmentation and flow cytometry
analyses (Figs. 2 and 3) indicated that at least
part of this decrease was due to apoptosis.
[View Larger Version of this Image (18K GIF file)]
Fig. 2.
The tadpole intestinal epithelial cells
but not the fibroblasts respond to T3 by undergoing programmed cell death. (A) T3-treatment of
epithelial cells resulted in the formation of a nucleosome-sized DNA ladder. The epithelial cells
were cultured on plastic dishes in the absence
(Control) or presence (T3 treated) of 100 nM T3 for 1 d. The genomic DNA was isolated,
electrophoresed on an agarose gel, stained with
ethidium bromide, and visualized under ultraviolet light. The DNA bands equivalent to the
lengths of the DNA in 1-4 nucleosomes were labeled on the right. (B) T3 induces dose-dependent DNA fragmentation in the tadpole intestinal epithelial cells. The epithelial cells were
cultured on plastic dishes in the presence of different concentrations of T3 for 3 d and then
DNA fragmentation was measured using the
ELISA methods. Note that DNA fragmentation was detectible with as low as 5-10 nM of T3, similar to that in the plasma during metamorphosis
(Leloup and Buscaglia, 1977), and plateaued at
100 nM T3. (C) Kinetics of T3-induced epithelial cell DNA fragmentation. The intestinal epithelial cells were cultured in the presence of 100 nM
T3 for 1-5 d and then DNA fragmentation was
analyzed with the ELISA method. Note that
DNA fragmentation reached the maximum after 3 or 4 d of treatment. (D) T3 induces DNA fragmentation in the epithelial cells but not
the fibroblasts. Intestinal epithelial cells and fibroblasts were isolated and cultured on 96-well plastic dishes (2 × 104 cells/well) for 1 or 3 d
in the presence or absence of 100 nM T3. DNA fragmentation was then determined by using the ELISA method.
[View Larger Versions of these Images (34 + 70K GIF file)]
Fig. 3.
Flow cytometry analysis indicates that epithelial cells
undergo apoptosis in response to T3 at different stages of the cell cycle. The epithelial cells were cultured in the presence or absence of 100 nM T3 for 2 or 3 d. The cells were then analyzed by
flow cytometry. Although the exact boundary between the live
cells and apoptotic cells (encircled area) was difficult to determine with precision, the results clearly showed that cells with different DNA contents or at different cell cycle stages (G2 at the
top and G1 at the bottom) were present in the apoptotic region
(reflected by the increased cellular granularity). Note that after 3 d
of treatment, essentially all cells were in the apoptotic region, and
were shown to be dead by trypan blue staining and DNA fragmentation (Figs. 1 and 2).
[View Larger Version of this Image (35K GIF file)]
) of T3 and the
maximal cell death at 100 nM of T3 (Fig. 2 B), in agreement
with the cell survival data above (Fig. 1 B). Kinetically, the
extent of DNA fragmentation was detectable by the ELISA
assay after 1 d of T3 treatment (Fig. 2 C), consistent with
the DNA fragmentation detected by the agarose gel assay
(Fig. 2 A). It continued to increase
4 or 5 d of T3 treatment (Fig. 2, C and D). In contrast to the epithelial cells,
the fibroblasts showed no detectable DNA fragmentation
above backgrounds at even 100 nM T3 (Fig. 2 D). Thus, T3
induces cell death specifically in the larval epithelium.
; Ishizuya-Oka and Shimozawa, 1987a
).
To investigate whether T3 causes apoptosis of the proliferating epithelial cells, we analyzed T3-treated primary cell
cultures using flow cytometry. The larval epithelial cells
cultured in the absence of T3 for 2 or 3 d had
10% of the
cells in the region of high granularity, i.e., apoptotic region
(Fig. 3, encircled area). In contrast, ~40 and 90% of the
cells were in the apoptotic region when treated with T3 for
2 and 3 d, respectively (Fig. 3), in agreement with the cell
survival analysis in Fig. 1 (B). Interestingly, the DNA content of the apoptotic cells ranged from subdiploid to nearly tetraploid (Fig. 3), suggesting that epithelial cells at different stages of the cell cycle, including the S- and G2-phases,
were susceptible to T3-induced cell death. The results further indicated that larval epithelial cells could proliferate
under the in vitro culture conditions, and that T3 did not
block this proliferation. Instead, T3 may induce both apoptosis and cell proliferation.
Fig. 4.
T3 stimulates the proliferation of both the intestinal epithelial cells and fibroblasts. Intestinal epithelial cells and fibroblasts were cultured overnight on 96-well plastic dishes (5 × 104
cells/well) in the presence or absence of 100 nM T3. 0.1 µCi of
[3H]thymidine was added to the 0.1-ml culture medium/well and
incubated for another 5 h. The amount of [3H]thymidine incorporated into genomic DNA was then measured.
[View Larger Version of this Image (73K GIF file)]
; Brown et al., 1996
; Gilbert et al.,
1996
). In Xenopus laevis intestine, >20 genes have been
shown to be regulated either directly or indirectly by T3
(Shi and Ishizuya-Oka, 1996
). Among them, IFABP (Ishizuya-Oka et al., 1994
; Shi and Hayes, 1994
) and a Na+/
PO43
cotransporter gene (Ishizuya-Oka et al., 1997
) have
been shown to be expressed in the intestinal epithelium.
To determine whether the T3-induced epithelial apoptosis
in vitro has a similar gene regulation profile as in tadpoles,
RNA was isolated from epithelial cells cultured for 1 d in
the presence or absence of 100 nM T3 and then analyzed
by Northern blot analysis.
). Similarly, the expression of Na+/PO43
cotransporter gene was drastically
downregulated after 1 d of T3 treatment (Fig. 5 B). The IFABP gene is known to be downregulated by T3 in tadpoles
(Shi and Hayes, 1994
). The Na+/PO43
cotransporter gene,
on the other hand, is known to be first upregulated and
then downregulated when premetamorphic tadpoles of
stage 56 or younger are treated with 5 nM T3 (Ishizuya-Oka et al., 1997
). Its mRNA level peaks around stages 58-60
in the intestine during normal development (Ishizuya-Oka
et al., 1997
). Thus, it is not surprising that T3 treatment of
intestinal epithelial cells from stage 57/58 tadpoles resulted in the downregulation of this gene (Fig. 5).
Fig. 5.
T3 treatment of intestinal epithelial cells leads
to the downregulation of
two known epithelial specific genes. (A) Kinetics of
the downregulation of IFABP gene by T3 in vitro. Epithelial cells from stage 57/58
tadpole intestine were cultured on plastic dishes in the
presence of 100 nM T3 for indicated numbers of hours and then RNA was isolated
for Northern blot analysis of
IFABP mRNA. (B) Regulation of IFABP and Na+/
PO43 cotransporter genes
by T3 in vitro. Epithelial cells
from stage 57/58 or stage 64 tadpoles were isolated and
cultured on plastic dishes in
the presence or absence of
100 nM T3 for 1 d. The RNA was isolated and analyzed by Northern blot hybridization with the cDNA probes for IFABP and intestinal Na+/PO43
cotransporter (NaPi). Note that both genes were
downregulated in the stage 57/58 epithelial cells as expected from
their expression during normal development (Shi and Hayes,
1994
; Ishizuya-Oka et al., 1994
, 1997). However, their downregulation in stage 64 epithelial cells appeared to contradict with expectation (see text for discussion). The hybridization with rpL8
served as a loading control.
[View Larger Versions of these Images (43 + 58K GIF file)]
cotransporter genes
was downregulated even when the intestinal epithelial cells
from stage 64 tadpoles were cultured in vitro in the presence of 100 nM T3 (Fig. 5 B). This contrasts with their upregulation during stages 62-64 when adult epithelial cells
differentiate in vivo (Shi and Hayes, 1994
; Ishizuya-Oka
et al., 1994
, 1997). As the epithelial cells at stage 64 are
adult type, they are capable of proliferating and differentiating even in the presence of T3 in intact tadpoles. The results here suggest that the epithelial cells dissociated from
the mesenchyme, and the basal lamina between the epithelium and the mesenchyme respond to T3 differently. Indeed, the epithelial cells from stage 64 tadpoles underwent
cell death just like those from stage 57/58 tadpoles in the
presence of T3 as assayed by DNA fragmentation (Fig. 6).
It is also interesting to note that cell death was induced by
T3 in spite of the copresence of epithelial and mesenchymal cells in vitro (Fig. 6), suggesting that the mere presence of nonepithelial components is not sufficient to prevent epithelial cell death.
Fig. 6.
Both larval and adult intestinal cells undergo apoptosis
upon T3 treatment in vitro. Cells were dissociated from intestine of stage 57/58 or 64 tadpoles and all of the cells were cultured together in vitro in the presence or absence of 100 nM T3. Cell death
was analyzed by using the DNA fragmentation ELISA assay. The
cells were predominantly epithelial but a higher portion of mesenchymal cells were present in stage 64 tadpole intestine (McAvoy and Dixon, 1977; Ishizuya-Oka and Shimozawa, 1987a
).
Note that cell death were detected for both the larval and adult
intestinal cells. Slightly lower levels of T3-induced DNA fragmentation were observed at stage 64, probably reflecting the presence
of a slightly higher percentage of nonepithelial cells.
[View Larger Version of this Image (28K GIF file)]
; Murata and
Merker, 1991
; Shi and Ishizuya-Oka, 1996
). Furthermore, the gene expression studies above suggest that dissociated
adult intestinal epithelial cells have a distinct response to
T3 than when they are in intact animals. Thus, it is likely
that ECM plays a role during intestinal remodeling. To investigate such a possibility, we cultured the larval epithelial cells on plastic dishes coated with various components
of the basal lamina (laminin, collagen IV, and fibronectin).
For a comparison, we also used dishes coated with collagen I, a major component of the connective tissue. Although the cells did not attach to plastic dishes, they attached well to all matrix-coated dishes (Fig. 7 and data not
shown). These coatings not only facilitated cell attachment
but also produced a more extended or spread-out cell
shape, whereas cells on the plastic dish were round. In
general, all coatings were found to enhance the epithelial
cell survival in vitro, increasing the survival time (when
95% of cells were dead) from ~1 wk on plastic dishes to
~2 wk on matrix-coated dishes.
Fig. 7.
Intestinal epithelial cells cultured on fibronectin-coated
dishes survive longer in the presence and absence of T3 than
those on plastic dishes. (A) The cells were cultured on the coated
dishes in the presence or absence of 100 nM T3 for 0 or 7 d and
then photographed. Note that the cells attached nicely to the
coated dishes in contrast to their behavior on plastic dishes, and
that some cells were still present after 7 d of T3 treatment, in contrast to those on plastic dishes (Fig. 1). (B) The cells were cultured on fibronectin-coated dishes in the presence or absence of
100 nM T3 and then the live cells were counted at different days
after trypsinization and trypan blue staining.
[View Larger Versions of these Images (12 + 97K GIF file)]
Fig. 8.
Epithelial cells from stage 57/58 intestine
cultured on matrix-coated dishes are more resistant to T3-induced apoptosis, but are similarly
stimulated by T3 to proliferate or downregulate IFABP expression. (A) Intestinal epithelial cells
were cultured on plastic dishes with or without indicated coatings for 1-3 d in the presence or absence of 100 nM T3. DNA fragmentation was then
measured by the ELISA method. (B) Epithelial
cells cultured on various matrix-coated dishes
have a similar dose response to T3 treatment in
spite of their increased resistance to T3-induced cell death. The epithelial cells were cultured on
various dishes in the presence of different concentrations of T3 for 3 d and then DNA fragmentation was then determined by the ELISA
method. (C) Epithelial cells were cultured overnight on various matrix-coated dishes in the presence or absence of 100 nM T3 (5 × 104
cells/well). 0.1 µCi of [3H]thymidine were added into the 1-ml culture medium/well and then incubated for another 5 h. [3H]thymidine
incorporated into genomic DNA was then measured. (D) Epithelial cells were cultured on matrix-coated dishes in the presence or absence of 100 nM T3 for 1 d and then total RNA was isolated for Northern blot analysis of IFABP mRNA. The hybridization with rpL8
served as a control.
[View Larger Versions of these Images (36 + 25 + 68 + 56K GIF file)]
; White,
1996
). In addition, the participation of signal transduction
pathways involving phosphatases/kinases has also been
suggested by the ability of immunosuppressants FK506
and cyclosporin A (CsA) to inhibit activation-induced T cell
death (Shi et al., 1989
; Birer et al., 1990
). To investigate
the possible involvement of similar pathways during amphibian metamorphosis, we tested the ability of four of
these inhibitors to block T3-induced intestinal epithelial cell
death. These include aurintricarboxylic acid (ATA; Shi et al.,
1994
) which is a nuclease inhibitor, Z-Val-Ala-Asp-floromethalketone (Z-VAD; Muzio et al., 1996
; Pronk et al.,
1996
), which is an ICE-like protease inhibitor, CsA, and
Fk506.
Fig. 9.
T3-induced intestinal epithelial cell death but not cell
proliferation can be inhibited by some but not all known inhibitors of mammalian apoptosis. (A) The epithelial cells were cultured on plastic dishes for 3 d in the presence or absence of 100 nM T3 and/or 300 ng/ml CsA, 10 ng/ml FK506 (FK), 100 µM ATA,
and 50 µM Z-VAD (VAD). DNA fragmentation was then measured by the ELISA method. Note that with the exception of
FK506, all inhibitors blocked T3-induced epithelial cell DNA
fragmentation. None of the drugs had any effect on DNA fragmentation by itself. (B) Time course of the drug inhibition of T3-induced epithelial cell death. Note that again with the exception
of FK506, all drugs inhibited cell death throughout the treatment.
The concentrations of the drugs used were 600 ng/ml CsA, 10 ng/ml
FK506 (FK), 100 µM ATA, and 50 µM Z-VAD (VAD), respectively. (C) CsA does not block T3-induced epithelial cell proliferation. The epithelial cells were cultured in the presence or absence
of 100 nM T3 and/or 600 ng/ml CsA for 1 d. Cell proliferation was
determined as in Fig. 4. (D) The downregulation of IFABP gene
in vitro by T3 is resistant to CsA and FK506. Epithelial cells from
stage 57/58 tadpoles were cultured on plastic dishes in the presence or absence of 100 nM T3 and/or 600 ng/ml CsA or 10 ng
FK506 (FK) for 1 d. The RNA was then isolated and analyzed as
above. Note that FK506 had no effect on either cell death (A and
B) or IFABP downregulation. Although CsA could inhibit cell death (A and B), it failed to block T3-induced IFABP gene regulation. The hybridization with rpL8 served as a loading control.
[View Larger Versions of these Images (50 + 22K GIF file)]
; Birer
et al., 1990
). Furthermore, both have been shown to exert their immunosuppressive effect by inhibiting calmodulin-dependent tyrosine phosphatase calcineurin (McKeon,
1991
; Schreiber, 1992
). This suggests that they may act at
an early step of the signal transduction pathway leading to
T cell death, in contrast to the inhibitors of ICE-like enzymes or nucleases. Thus, we treated epithelial cells from
stage 57/58 tadpole intestine with or without T3 in the
presence or absence of CsA and FK506, and then analyzed the effects of such treatment on IFABP expression. Again,
T3 treatment alone led to the downregulation of the gene
(Fig. 9 D). In agreement with its inability to block the T3-induced apoptosis, FK506 had no effect on the expression
of the IFABP gene (Fig. 9 D). Interestingly, under the conditions used, CsA could completely block the T3-induced cell death, but it could not prevent the downregulation of
the IFABP gene (Fig. 9 D). Thus, whereas the IFABP
gene downregulation does not appear to be a direct effect
of T3 (Shi and Hayes, 1994
), it may still precede the step of
the cell death pathway that is sensitive to CsA inhibition.
Alternately and more likely, T3-induced downregulation
of the IFABP gene is in a parallel pathway independent of
the cell death pathway. Thus, CsA can block T3-induced
apoptotic pathways but not the T3-induced cell proliferation or regulation of some T3 response genes.
Discussion
; Tata et al., 1991
). In the frog intestine, two major cell
types exist, epithelial and fibroblasts (Ishizuya-Oka and
Shimozawa, 1987a
). While the fibroblasts rapidly proliferate and differentiate during metamorphosis (Ishizuya-Oka and Shimozawa, 1987a
), the larval epithelial cells undergo
degeneration through an apoptotic process (McAvoy and
Dixon, 1977
; Ishizuya-Oka and Shimozawa, 1992a
). Our
results indicate that at least part of the intestinal remodeling, i.e., the epithelial cell death, can be reproduced in primary cultures of separated cells in vitro in the presence of
T3, suggesting that the apoptotic event is cell autonomous.
Furthermore, this T3-dependent apoptosis has the same
cell type specificity as in vivo (McAvoy and Dixon, 1977
; Ishizuya-Oka and Shimozawa, 1992a
, b
).
, 1992a
, b
). Interestingly,
T3 treatment of the fibroblast in vitro leads to an increase
in cell proliferation, suggesting that the development of
the fibroblasts during metamorphosis is through the action
of T3 on those cells directly, i.e., cell autonomous.
,
1978
; Ishizuya-Oka and Shimozawa, 1987a
). Thus, T3 may
control a common set of genes present in both the epithelial cells and fibroblasts of the intestine that can facilitate
the cell proliferation. What separates the larval epithelial
cells from the other major intestinal cells, the fibroblasts, is their apoptotic response to T3. This latter response occurs in epithelial cells at all stages of the cell cycles. The final outcome of the T3 treatment is the total degeneration
of the larval epithelial cells both in vivo and in primary cell
cultures. This differential effect of T3 on epithelial cells
and immature fibroblasts suggests that T3 has the ability to
stimulate cell cycle progression, which leads to cell proliferation in non- or less-differentiated cells such as the fibroblasts, or to apoptosis in differentiated cells, such as
the epithelial cells.
; Ishizuya-Oka and Shimozawa, 1992a
). Our studies have provided biochemical and cell biological evidence for the
programmed cell death via apoptosis in the tadpole intestine.
), none of them correspond to known cell death or survival genes. However,
our inhibition studies clearly indicate the involvement of
ICE-like proteases and nucleases, just as in mammalian
apoptotic processes (Martin and Green, 1995
; White, 1996
).
Furthermore, the T3-induced epithelial cell death has a typical nucleosomal ladder of DNA fragmentation. Thus,
the cell death during the T3-dependent amphibian developmental process possesses many of the characteristics of
mammalian apoptotic model systems.
; Birer et al., 1990
). On the other hand,
FK506, which inhibits activation-induced T cell death (Birer et al., 1990
), has no effect on T3-induced intestinal epithelial apoptosis. The exact mechanisms by which CsA
and FK506 inhibit T cell death are unknown. However, both CsA and FK506 have been shown to be capable of inhibiting calmodulin-dependent phosphatase, and this inhibition has been suggested to be responsible for their effects in T cell death (Shi et al., 1989
). Our studies suggest
that such a mechanism may not be responsible for the inhibition of T3-induced intestinal cell death by CsA.
). Nur77 is required for T cell death, and belongs to the superfamily of nuclear hormone receptors
that also include TRs (Liu et al., 1994
; Woronicz et al.,
1994
; Mangelsdorf et al., 1995
). It is, therefore, suggested that the inhibition of Nur77 activity by CsA may block the
ability of Nur77 to regulate its target genes, thus preventing activation-induced T cell death. As TRs and Nur77 belong to the same receptor family and share many functional features, it is possible that CsA may inhibit TR
function, thus blocking the intestinal epithelial cell death.
However, our results on the expression of the IFABP gene clearly rule out such a mechanism, as the IFABP gene was
downregulated by T3 both in the presence or absence of
CsA. Thus, CsA functions either in a parallel pathway independent of the pathway leading to IFABP gene regulation or downstream of the IFABP gene regulation.
; Timpl and Brown, 1996
). The
ECM serves as a structural support for the cells it surrounds and is essential for the integrity and morphology of
an organ. Equally as important, ECM can modulate a number of cellular functions, such as cell migration, morphology, proliferation, differentiation, and death (Hay, 1991
;
Schmidt et al., 1993
; Ruoslahti and Reed, 1994
).
; Simon-Assmann and
Kedinger, 1993
; Shi and Ishizuya-Oka, 1996
). During amphibian metamorphosis, extensive remodeling of the intestinal basal lamina (Ishizuya-Oka and Shimozawa, 1987b
;
Murata and Merker, 1991
) has been observed to coincide with frequent migration of macrophages across the lamina
into degenerating larval epithelium and extensive direct
contacts between the developing adult epithelial cells and
mesenchyme (Ishizuya-Oka and Shimozawa, 1987b
, 1992b
).
; Brown et al., 1996
; Stolow
et al., 1996
). This family of Zn-dependent extracellular enzymes are capable of digesting various components of the
ECM (Alexander and Werb, 1991
; Matrisian, 1992
; Birkedal-Hansen et al., 1993
; Sang and Douglas, 1996
). Of particular interest is stromelysin-3, whose substrates in the ECM
remain to be identified. This gene has been found to be activated in different organs immediately before and during
cell death (Patterton et al., 1995
; Brown et al., 1996
). More
importantly, its spatial and temporal expression correlates
precisely with the basal lamina modification in the intestine as summarized above (Ishizuya-Oka et al., 1996
). In
contrast, the collagenase-3, collagenase-4, and gelatinase
A are either minimally regulated or activated only during
or toward the end of intestinal epithelial degeneration (Patterton et al., 1995
; Stolow et al., 1996
). These results argue for a role of specific modification of the basal lamina by
metalloproteinases during T3-induced epithelial apoptosis.
In support of this, a number of metalloproteinase genes
are also activated during apoptotic degeneration of the
postlactation mammary gland (Talhouk et al., 1992
; Lund
et al., 1996
); and overexpression of stromelysin-1 in the
mammary gland leads to matrix modification and apoptosis (Witty et al., 1995
; Alexander et al., 1996
).
genes are reactivated in the adult epithelial cells as they differentiate in the presence of T3 (stages
62-66) (Shi and Hayes, 1994
; Ishizuya-Oka et al., 1994
,
1997). However, when epithelial cells isolated from stage
64 tadpoles were cultured in vitro, T3 treatment led to the
downregulation of these genes, just like the cells isolated from premetamorphic tadpoles. Thus, the removal of the
ECM and the underlying mesenchyme rendered the adult
epithelial cells at stage 64 to undergo apoptosis in response to T3. Although individual ECM components fail
to prevent the downregulation of IFABP gene by T3, multiple interactions between epithelial cells and ECM in vivo
may be required for proper gene expression in epithelial cells. Alternatively, epithelial-mesenchymal interactions may also play an important role in adult epithelial development
as first suggested by the organ culture experiments (Ishizuya-Oka and Shimozawa, 1992b
). However, coculturing
mesenchymal and epithelial cells fails to prevent cell death
(Fig. 6). Thus, both cell-cell and cell-ECM interactions
are likely to be important for adult epithelial development.
Our results further suggest that the differentiated intestinal epithelial cells are intrinsically vulnerable to T3-induced death. What prevents the adult epithelial cells from T3-
induced apoptosis is partially because of the new ECM-
epithelial and/or mesenchymal-epithelial interactions established during metamorphosis. Such a conclusion is also
consistent with the self-renewal of adult intestinal epithelium during which epithelial cells gradually migrate as they
differentiate toward the crest of the fold, equivalent to
mammalian intestinal villus (Shi and Ishizuya-Oka, 1996
).
After a finite period of time, the cells at the crest but not
elsewhere undergo apoptosis, partially because of altered
cell-cell and cell-ECM interactions, and are replaced by
the newly arrived epithelial cells (McAvoy and Dixon, 1977
;
Ishizuya-Oka and Ueda, 1996
).
; Damsky and Werb, 1992
; Montgomery et al., 1994
; Ruoslahti
and Reed, 1994
; Boudreau et al., 1995
; Brown and Yamada, 1995
). In one of the best studied model systems, i.e., the development of the mammary gland, it has been proposed that the interaction of ECM with its integrin receptors leads to the activation of focal adhesion tyrosine kinase, which in turn transduces the signal through the
mitogen-activated kinase pathway to the nucleus (Roskelley et al., 1995
). This or similar mechanisms may be responsible for ECM-mediated transcriptional regulation of gene expression (Roskelley et al., 1994
). Such ECM-mediated gene expression may also play a role in regulating the
fate of intestinal epithelial cells during T3-dependent metamorphosis.
Received for publication 16 May 1997 and in revised form 22 August 1997.
M.A. Stolow's present address is OriGene Technologies, Inc., 13 Taft Court, Suite 111, Rockville, MD 20855.We would like to thank P. Henkart, H. Kleinman, and K. Yamada for helpful discussions and suggestions, and T. Vo (all from National Institutes of Health) for preparation of the manuscript.
CsA, cyclosporin A;
ECM, extracellular matrix;
ICE, interleukin-1-converting enzyme;
IFABP, intestinal
fatty acid binding protein;
T3, thyroid hormone or 3,5,3
-triiodothryonine;
TR, thyroid hormone receptor;
Z-VAD, Z-Val-Ala-Asp-floromethalketone.
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