The endoplasmic reticulum stress transducer OASIS is involved in theterminal differentiation of goblet cells in the large intestine
Rie Asada, Atsushi Saito, Noritaka Kawasaki, Soshi Kanemoto, Hideo Iwamoto, Mami Oki,
Hidetaka Miyagi, Sotaro Izumi, and Kazunori Imaizumi*
Department of Biochemistry, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3
Kasumi, Minami-ku, Hiroshima 734-8551, Japan
Running title: Physiological UPR during goblet cell maturation*Address correspondence to: Kazunori Imaizumi, Department of Biochemistry, Graduate School of
Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan
Tel: +81-82-257-5130, FAX: +81-82-257-5134, E-Mail: imaizumi@hiroshima-u.ac.jp

Background: OASIS is an ER stress sensor
expressed in the large intestine.
Results: Mature goblet cells are decreased in
Oasis-/- mice. Knockdown of the Oasis
transcript impairs the maturation of goblet
cells.
Conclusion: OASIS plays crucial roles interminal differentiation of goblet cells.
Significance: OASIS signaling during the ER
stress response is involved in celldifferentiation and maturation.

changes and the expression of goblet celldifferentiation markers in the large intestineof Oasis-/- mice. By disrupting the Oasis
gene, the number of goblet cells andproduction of mucus were decreased in thelarge intestine. Oasis-/- goblet cells showedabnormal morphology of mucous vesicles
and rough ER. The expression levels of
mature goblet cell markers were lower, andconversely those of early goblet cell markers
were higher in Oasis-/- mice, indicating thatdifferentiation from early to mature goblet
cells is impaired in Oasis-/- mice. To
determine the association of OASIS withother factors involved in goblet celldifferentiation, in vitro experiments using
cell culture model were performed. We
found that OASIS was activated in responseto mild ER stress which is induced indifferentiating goblet cells. Knockdown of
the Oasis transcript perturbed goblet cell
terminal differentiation. Together, our data
indicate that OASIS plays crucial roles inpromoting the differentiation of early goblet
cells to mature goblet cells in the large
intestine.

OASIS is a bZIP transmembranetranscription factor localized in theendoplasmic reticulum (ER) that is cleaved
in its transmembrane region in response to
ER stress. This novel ER stress transducer
has been demonstrated to express inosteoblasts and astrocytes and promoteterminal maturation of these cells.
Additionally, OASIS is highly expressed ingoblet cells of the large intestine. In thisstudy, we investigated the roles of OASIS ingoblet cell differentiation in the large
intestine. To analyze the functions of OASISin goblet cells, we examined morphological1

astrocytes of the central nervous system(20-22). From the analysis of knockout mice,
OASIS has been demonstrated to be involvedin terminal differentiation and osteoblasts(23-25) and astrocytes (submitted by Saito).
The intestinal epithelium is composed offour distinct cell types, including the absorptive
enterocytes and the goblet, Paneth andenteroendocrine secretory cell lineages (26, 27).
Stem cells are committed to generate theselineages by the Wnt and Notch signalingcascades: Wnt signaling is required for thegeneration of the secretory lineages, whereas
Notch signaling is necessary for thedifferentiation of enterocytes (28, 29).
However,themolecular
mechanisms
underlying the differentiation of intestinal
epithelial cells are incompletely defined.
Recently,
the
ETS
(E26
transformation-specific)-domain transcription
factor,SPDEF(SAM-pointed
domain-containing ETS-like factor) (30, 31),
has been reported to act downstream of
ATOH1 (32), which is an essential determinantof secretory lineages downstream of β-catenin(33, 34). Maturation of Paneth and goblet cells
has been shown to be impaired in Spdef
deficient mice, whereas immature secretory
progenitors accumulate in the intestine (35).
These observations suggest that SPDEF
promotes the terminal differentiation of asecretory progenitor pool into Paneth andgoblet cells. Interestingly, the expression of
CREB4, an ER stress transducer that is
structurally similar to OASIS, is completely
abolished in Spdef-deficient Paneth and goblet
cells (35). These observations suggest that theexpression of CREB4 is controlled atdownstream of SPDEF, and that ER stressresponse signaling through ER stresstransducers such as CREB4 mediates thedifferentiation and maturation of secretory celllineages in the intestinal epithelium. In thisstudy, we investigated the roles of the ER
stress transducer OASIS, which is expressed in
the crypt base of the large intestinal epithelium,in the differentiation of goblet cells.

The endoplasmic reticulum (ER) is acentral cellular organelle responsible for thesynthesis, folding and posttranslational
modifications of proteins destined for thesecretory pathway. A number of cellular stressconditions lead to the accumulation of
unfolded or misfolded proteins in the ER
lumen. These conditions, which arecollectively termed ER stress, have thepotential to induce cellular damage (1, 2). The
ER responds to these perturbations byactivating an integrated signal transduction
pathway through the ER stress transducers,which is called the unfolded protein response(UPR) (3-5). The UPR involves at least three
distinctcomponents;
1)translational
attenuation to decrease the demands made on
the organelle (6); 2) transcriptional inductionof genes encoding ER-resident chaperones tofacilitate protein folding (7, 8); and 3)
ER-associated degradation (ERAD) to degrade
the unfolded proteins accumulated in the ER (9,10). If these strategies fail, cells undergo ER
stress-induced apoptosis (11, 12). The UPR
was originally described as a system by whichcells evade damage in response to acute ER
perturbation. However, recent advances haverevealed that the UPR also provides importantsignals for regulating cell differentiation andmaturation, or the maintenance of basal cellular
homeostasis (13-16).
Previously, we identified OASIS (oldastrocyte specifically induced substance) as anovel ER stress transducer (17). OASIS isbasic leucine zipper (bZIP) transcription factor
that belongs to the cyclic AMP-responsive
element (CRE)-binding protein/activatingtranscription factor (ATF) family. Although
OASIS is localized to the ER membrane undernormal conditions, it is cleaved at themembrane in response to ER stress.
Consequently,
itscleavedN-terminal
cytoplasmic domain, which contains the bZIP
domain, translocates into the nucleus where it
activates the transcription of target genes (18,19). High expression of OASIS was observedin the osteoblasts of osseous tissues and the2Consequently, we demonstrated that OASIS
plays crucial roles in promoting the terminal
differentiation of goblet cells in the large
intestine.

RNA isolation and RT-PCR − Total RNA was
isolated from the large intestine of 3-week old
mice or LS174T cells using ISOGEN (Wako)
according to the manufacturer's protocol.
First-strand cDNA was synthesized in a 20 μl
reaction volume using a random primer(Takara) and Moloney murine leukemia virus
reverse transcriptase (Invitrogen). PCR was
performed using each specific primer set in a
total volume of 30 μl containing 0.8 μM ofeach primer, 0.2 mM dNTPs, 3 units of Taqpolymerase, and 10× PCR buffer (Agilent).
Primer sequences are summarized in
Supplementary Information, Table S1. The
PCR products were resolved by electrophoresis
on a 4.8% acrylamide gel. The density of eachband was quantified using the Adobe
Photoshop Elements 2.0 Program (Adobe
Systems Incorporated).

EXPERIMENTAL PROCEDURES
Animals – 3-week old C57BL/6 mice orOasis-/- mice were used in this study. The
Oasis-/- mice were previously established in
our laboratory (22-24). In all studies comparing
wild-type and Oasis-/- mice, sex-matchedlittermates derived from the mating of Oasis+/mice were used. The experimental proceduresand housing conditions for animals wereapproved by the Committee of Animal
Experimentation, Hiroshima University.
Cell culture, treatments and transfection −LS174T cells were cultured in Eagle's minimal
essential medium supplemented with 10% FCS
and 1% non-essential amino acids. LS174T
cells were cultured overnight in 9.2 cm2 dishfor RNA or protein isolation, and in a Lab–Tek
Chamber Slide (Nalge Nunc) for periodic
acid-Schiff (PAS) staining. Subsequently, cells
were treated with 2 mM sodium butyrate,
which induces LS174T cells to differentiate tomature goblet cells. For Oasis knockdown
experiments, LS174T cells were transfectedwith 1 μg Oasis siRNA (predesigned siRNApooltargetingOasis:AAAAGAAGGUGGAGACAUU,GGGACCACCUGCAGCAUGA,GAAGGAGUAUGUGGAGUGU,andCAGGAGAGCCGUCGUAAGA;
ThermoScientific Dermacon, M-008579-01-0005) orcontrol siRNA (Silencer Cy3 labeled negative
control No.1 siRNA; Life Technologies,
AM4621). Transfection was performed 12 hrs
before treatment with sodium butyrate using
Lipofectamine
2000
reagent(Life
Technologies) according to the manufacturer’s
protocols. 24 hrs after treatment with sodium
butyrate, PAS staining was performed as
described previously (36). The PAS-stained
slide was counterstained with hematoxylin
solution.

Western blotting − For western blotting,proteins were extracted from LS174T cells
using cell extraction buffer containing 10%
SDS, 0.5 M EDTA (pH 8.0), 100 mM
methionine, and a protease inhibitor mixture(MBL International). The lysates wereincubated on ice for 45 min. Aftercentrifugation at 16,000 × g for 15 min, theprotein concentrations of the supernatants were
determined. Protein-equivalent samples were
loadedonto
sodiumdodecylsulfate-polyacrylamidegels.
Anti-β-actin(Sigma; 1:3000) and anti-OASIS (purified
from a hybridoma as described previously (22,23)) antibodies were used for western blotting.
The density of each band was quantified using
the Adobe Photoshop Elements 2.0 Program(Adobe Systems Incorporated).
Histological analysis and in situ hybridization− Large intestine from 3-week old mice was
fixed overnight in 10% Formalin Neutral
Buffer Solution (Wako). Samples were thendehydrated with ethanol, embedded in paraffinand sectioned (5 μm). Hematoxylin-eosin
staining and PAS staining were performed3

according to standard protocols. In situ
hybridization
was
performed
using
digoxigenin-labeledcRNA
probes(Supplementary information, Table S2).
Antisense and sense probes were made by invitro transcription in the presence ofdigoxigenin-labeled dUTP, using various
cDNAs subcloned into the pGEM-Teasy vector(Promega) as templates. Large intestine
isolated for in situ hybridization was frozen
immediately and sectioned (6 μm). The frozen
sections were fixed for 20 min with 4%
formalin in phosphate-buffered saline (PBS,
pH 7.4). The sections were then washed with
PBS and treated with 0.1% proteinase K for 5
min. After washing with PBS, the sections
were re-fixed for 20 min with 4% formalin in
PBS, and treated with 0.1M triethanolamine,
2.5% anhydrous acetic acid for 10 min,followed by washing with PBS. Sections wereprehybridized for 1 hr at 37°C in hybridizationbuffer (0.01% Dextran Sulfate, 0.01M
Tris-HCl pH 8.0, 0.05 M NaCl, 50%
Formamide, 0.2% Sarcosyl, 1× Denhardt’s
solution, 0.5 mg/ml Yeast tRNA, 0.2 mg/ml
Salmon testis DNA), and then hybridized
overnight at 55°C in hybridization solution
with 100 ng/ml cRNA probe. After washingwith 4× saline sodium citrate buffer for 20 min
at 60°C, the sections were further washed 2×
saline sodium citrate buffer, 50% formamide
for 30 min at 60°C. Sections were treated
RNaseA in RNase buffer (10 mM Tris-HCl
pH7.4, 1 mM 0.5 M EDTA (pH 8.0), 0.5 M
NaCl) for 30 min at 37°C to remove
un-hybridized probe. After RNase treatment,
sections were washed with 2× saline sodium
citrate buffer, 50% formamide for 30 min at
60°C, and then blocked with 1.5% blocking
reagent in 100 mM Tris-HCl pH 7.5, 150 mM
NaCl for 1 hr at room temperature. For
detection of digoxigenin-labeled cRNA probes,
anti-digoxigenin antibody conjugated toalkaline phosphatase was used at a dilution of
1:500, and color was developed by incubation
with 4-nitro blue tetrazolium chloride and5-bromo-4-chloro-3-indolyl
phosphatesolution.
Electron microscopy − Large intestine from
3-week old mice was fixed in 1%
glutaraldehyde in PBS for 15 min. Afterwashing with distilled water, the tissues were
post-fixed in 0.5% osmium tetroxide in 0.1 M
cacodylate buffer for 30 min. Following
dehydration, the tissues were embedded in
EPON812, and ultra-thin sections were stainedwith uranyl acetate and lead citrate. Stained
sections were visualized using a Hitachi 7100
electron microscope operated at 80 kV. The
mean cell area was determined using ImageJsoftware (NIH).
Statistical analysis − Statistical comparisonswere made using the unpaired-Student’s-t-test.
Statistical significance between two samples
was determined by a P value of less than 0.05.
P values of less than 0.05, 0.01, or 0.001 aredescribed as *P < 0.05, **P < 0.01, or***P<0.001, respectively.
RESULTSOASIS was highly expressed in immaturegoblet cells of the large intestine − Wepreviously reported that OASIS is expressed inosteoblasts and astrocytes (17, 20, 21, 23). To
examine the tissue distribution of Oasis mRNA
more precisely, we performed RT-PCR usingmRNA isolated from various tissues of 3-week
old mice. We detected strong Oasis mRNAsignals in submandibular gland, lung, stomach,and in the large intestine where it was mostintense (Fig.1A). Oasis mRNA was expressed
highly in all portions of the large intestine
except for the appendix. In contrast, the levelsof Oasis mRNA were very low in the small
intestine (Fig.1B). In the digestive tract, there
are three distinct cell types, i.e. absorptive
enterocytes, enteroendocrine cells, and goblet
cells. To identify which of these cells
expressed Oasis mRNA, we carried out in situ
hybridization using Oasis cRNA probes. The

4

Oasis signals were focally detected in the baseof the crypt, but not in the apical portion of thecrypt (Fig.1C). In contrast, Gapdh mRNA wasobserved in all cells (both basal and apical) of
the crypt. The cells expressing Oasis mRNA
possessed vacuoles in their cytosol, indicatingthat OASIS is expressed in goblet cells.
Moreover, the goblet cells at the base of thecrypt are immature cells that are developingfrom intestinal stem cells (27, 37, 38). Thus,
we concluded that the cells expressing OASIS
were immature goblet cells.

abnormally expanded in these cells (Fig.3D).
In the upper portion of the crypt, cells withabnormal ultrastructure were also observed(Fig.3F). The other types of cells in the crypt
epithelium, such as enteroendocrine cells andenterocytes, were morphologically intact inOasis-/- mice (data not shown). Goblet cells
are known to be differentiated from intestinal
stem cells in the crypt base, and the maturegoblet cells move to the apical portion of thecrypt. We observed abnormal morphology ofgoblet cells from the base to the apical portionof the crypt in Oasis-/- mice, suggesting that
deletion of the Oasis gene affected thedifferentiation or maturation of goblet cells
from the intestinal cell lineage involving
impairment of vesicle formation.

The numbers of mature goblet cells
decreased in Oasis-/- mice − To elucidate thefunctions of OASIS in immature goblet cells,
we first performed histological analysis in the
Oasis-/- mice that we generated previously(22-24). The mice were born at the expected
Mendelian ratios and were fertile, but showed
impaired bone formation. However, the generalmorphology of all other tissues and organs was
normal, and the architecture of the intestinal
tract was unaffected. Additionally, formationof the crypt and differentiation of enterocytes
also appeared normal. However, the apparent
numbers of goblet cells that contained
abundant vacuoles were decreased in Oasis-/mice (Fig.2A). PAS staining showed a marked
decrease in the number of cells containing
mucus both in the proximal and distal large
intestine of Oasis-/- mice (Fig.2B-E). In
contrast, immature cells that contain lessmucus were increased in the crypt epithelium,
suggesting that the differentiation or
maturation of goblet cells was inhibited in
Oasis-/- mice.
Next, we carried out electron microscopic
examination of goblet cells in WT and Oasis-/mice. Normal goblet cells have abundantcytoplasm containing large secretory vesicles(Fig.3A). In Oasis-/- mice, almost all goblet
cells at the crypt base and in the lower portionof the crypt showed an abnormal ultrastructurein which both the number and size of vesicles
were decreased, and the membranes of somevesicles were often fused (Fig.3B). Moreover,
the rough endoplasmic reticulum was

OASIS
functionsintheterminaldifferentiation of goblet cells − We next
focused on the roles of OASIS in goblet celldifferentiation. Intestinal epithelial stem cells
are determined to become cells of the secretory
lineagesuch
asgobletcells
andenteroendocrine cells by Notch ligand
Delta-like 1 (Dll1)(39), whereas absorptive
enterocytes are specified by Notch and thetranscription factor Hairy and enhancer of split
1 (HES1) (40). Subsequently, secretory
progenitor cells are specified to become goblet
cells by the transcription factors, Atonal
homolog 1 (ATOH1) (33, 34) and Growth
factor independent 1 (GFI1) (41, 42). We
examined the expression levels of various
differentiation markers of the goblet celllineages in the large intestine of Oasis-/- miceby RT-PCR. All genes involved in the primarydifferentiation from stem cells to early goblet
cells or absorptive enterocytes were normally
expressed in Oasis -/- mice (Fig.4A). Therefore,
intestinal epithelial stem cells are correctly
specified to both the goblet cell and absorptive
enterocyte
lineages.
Interestingly,
theexpression of Trefoil factor 3 (Tff3), an earlygoblet cell marker, was significantly increasedin Oasis-/- mice (Fig.4A). Conversely, themature goblet cell markers, Muchin 2 (Muc2),
Anterior gradient 2 (Agr2), and Resistin like5beta (Retnlb), were markedly decreased(Fig.4B). In situ hybridization revealed Tff3mRNA signals were observed diffusely in thecrypt: the intensity was very faint in WT mice,but elevated in Oasis-/- mice (Fig.4D). Muc2
mRNA signals were robustly observed
throughout the crypt epithelium in WT mice,
but were almost abolished in Oasis-/- mice
(Fig.4E). These
resultssuggestthat
differentiation from early to mature goblet cells
is impaired in the large intestine of Oasis-/mice.
Recently, the Ets-domain transcription factor,
SPDEF, was reported to be required for theterminal differentiation of goblet cells (35). In
the large intestine, Spdef-/- mice showed asimilar phenotype to that of Oasis-/- mice, i.e.
goblet cells are correctly specified but theterminal differentiation of early to maturegoblet cells was impaired. CREB4 is an ER
resident CREB/ATF family transcription factor
that is structurally very similar to OASIS.
Interestingly, the expression of CREB4 wasreported to be down-regulated in the intestineof Spdef-/- mice, indicating that CREB4 could
be a direct target of SPDEF. We therefore
examined whether the SPDEF-CREB4
pathway is related to the OASIS signaling
pathway. The expression of Spdef mRNA was
not altered in the large intestine of Oasis -/mice (Fig.4C and 4F), implying that OASIS
does not regulate the expression of the Spdef
gene. In contrast, the expression of CREB4
was significantly decreased in Oasis -/- mice
(Fig.4C). These data suggest that theexpression of CREB4 is regulated by OASIS.
Combined with the published data, CREB4 istherefore a common downstream target of
OASIS and SPDEF, but the expression of
CREB4
isregulated
independently.
Furthermore, CREB4 may be a marker for theterminal differentiation of goblet cells, andmay function together with the OASIS
signaling pathway in the maturation of goblet
cells.

the roles of OASIS in the terminal
differentiation of goblet cells, we examined theexpression and activation of OASIS in thehuman colon cancer LS174T cell line. LS174T
cells are secretory progenitors that can be
induced to differentiate into goblet cells bytreatment with sodium butyrate (36). In
LS174T cells treated with 2 mM sodium
butyrate for 24 hrs, PAS-positive secretory
granules were produced (Fig. 5A), and theexpression of mature goblet cell markers suchas Muc2 and Agr2 was gradually induced from
6 hrs after treatment (Fig.5B). Western blotting
showed that the expression of full-length
OASIS increased after 6 hrs treatment withsodium butyrate, peaking at 12 hrs following
which it gradually decreased. The cleaved
fragment of OASIS, p50 OASIS (OASIS
N-terminus), also increased in synchrony withthe expression of full-length OASIS, indicatingthat OASIS is activated during thedifferentiation of LS174T cells to maturegoblet cells (Fig.5C). Because OASIS is
activated in response to ER stress (17), it ispossible that ER stress occurred during goblet
cell differentiation. We examined theexpression of ER stress markers after treatment
with sodium butyrate. Messenger RNAs for the
ER stress markers, Bip and Chop, were slightly
but significantly up-regulated in accordancewith the pattern of OASIS activation (Fig.5D).
These findings suggest that mild ER stress is
induced during goblet cell differentiation, andthat OASIS is subjected to regulated
intramembrane proteolysis in response to themild ER stress. When differentiating into
mature goblet cells, early goblet cells gradually
start to produce abundant proteins such as
MUC2, and these proteins are overloaded into
the ER. Thus, mild ER stress during maturationof goblet cells could be derived from a highdemand for synthesis and secretion of mucus
materials.
Our findings in Oasis -/- mice describedabove suggest that CREB4 could be expressed
as a common downstream target of OASIS andSPDEF during differentiation of early tomature goblet cells. We therefore examined theActivation of OASIS during terminal
differentiation of goblet cells − To investigate

6expression of Creb4 and Spdef mRNAs after
treatment of LS174T cells with sodium
butyrate. The expression of Creb4 mRNA was
induced 6 hrs after treatment, the same time
point at which expression of p50 OASIS(OASIS N-terminus) began (Fig.5E). Creb4mRNA expression then gradually decreased
from 12 hrs after treatment. In contrast,
although Spdef was also induced from 6 hrs,
the expression level was maintained until 24
hrs after treatment (Fig.5E), indicating that
SPDEF is not downstream of OASIS.

LS174T cells to mature goblet cells (Fig.6E).
However, the slight elevation of these genes
was not affected by knockdown of Oasis
mRNA, indicating that mild ER stress occurs
prior to OASIS activation during thematuration of goblet cells. Taken together, ourdata suggest that OASIS plays an essential rolein goblet cell differentiation following its
activation by mild ER stress (Fig.7).
DISCUSSION
We previously demonstrated that OASIS
promotes the differentiation of osteoblasts andis involved in bone formation (23-25). In thisstudy, we found that Oasis is highly expressedin goblet cells at the crypt base in the large
intestine. Goblet cells synthesize and secrete
large amounts of mucus proteins, including
mucin, and are similar to osteoblasts in thatabundant proteins are loaded into the ER.
Therefore, ER stress is easily induced insecretory goblet cells (43, 44). However,immature goblet cells do not produce mucus
and do not undergo ER stress. During
differentiation from secretory progenitor cells
to mature goblet cells, immature cells develop
the machinery for dealing with abundant
proteins in the ER, including the UPR system.
We did in fact demonstrate that ER stress is
induced during goblet cell differentiation, andis followed by the induction of genes expressedin mature goblet cells. Thus, it is possible that
ER stress is a trigger for the maturation ofgoblet cells. Similar phenomena are also seenin plasma cell differentiation. For the fulldevelopment of antibody secretion, activationof the UPR is essential (45, 46). Taken together,previous data and our findings suggest that
activation of the UPR by ER stress is required
for the maturation of immature cells to“professional” secretory cells.

OASIS is required for the differentiation ofgoblet cells − To determine whether OASIS isindispensable for the differentiation of goblet
cells, we examined the effects of Oasis
knockdown using Oasis siRNA on the
differentiation of LS174T cells. We confirmed
that
Oasis
siRNA
successfully
andsignificantly suppressed the expression of
OASIS in LS174T cells (Fig.6A). PAS-staining
showed that secretory granules weresignificantly decreased in LS174T cells
transfected with Oasis siRNA following
treatment with sodium butyrate (Fig.6B).
Furthermore, the mature goblet cell markers,
Muc2
and
Agr2,
were
dramatically
down-regulated (Fig.6C). These data suggestthat inhibition of OASIS expression impaired
the terminal maturation of LS174T cells togoblet cells.
Next, to analyze the relationship between
the OASIS and SPDEF pathways, we checked
the expression of SPDEF and CREB4 in
LS174T cells treated with sodium butyrate.
Even when the expression of OASIS was
suppressed by siRNA, the up-regulation of
Spdef mRNA following treatment with sodium
butyrate was not affected. However, inductionof CREB4 by sodium butyrate wassignificantly inhibited by transfection of
LS174T cells with Oasis siRNA (Fig.6D).
These findings support our hypothesis that
CREB4, but not SPDEF, is downstream of
OASIS.
The expression of ER stress markers was
slightly up-regulated during differentiation of

In the large intestine of Oasis-/- mice, thenumber of mature goblet cells that containabundant secretory granules was dramaticallyreduced. We found that Tff3, an early goblet

7

molecular mechanisms involved in goblet celldifferentiation.

cell marker, was up-regulated, while maturegoblet cell markers such as Muc2, Agr2, andRetnlb, were conversely down-regulated.
Genes involved in the primary differentiationof immature goblet cells were not affected,
indicating that intestinal stem cells arecorrectly specified to both secretory
progenitors and absorptive enterocyte lineages.
Thus, we concluded that the abnormalarchitectures observed in the crypt epitheliumof Oasis-/- mice are caused by impaired
terminal maturation of goblet cells. The
anomalies in the large intestine of Oasis-/mice are also reminiscent of the phenotype
seen in Spdef-/- mice, which showed aprofound loss of mature goblet cells (35). In
this study, the authors used microarray
analyses to show that OASIS is not
down-regulated to any extent in Spdef -/- colon.
Furthermore, we observed that the expressionof SPDEF was not altered in the large intestineof Oasis-/- mice. These findings suggest thatboth genes are involved in the terminal
differentiation of goblet cells, but by
independent pathways. Interestingly, theexpression of CREB4 was significantly
down-regulated both in Oasis-/- and Spdef-/large intestine, indicating that CREB4 is acommon downstream target of OASIS andSPDEF that may be crucial for the terminal
differentiation of goblet cells (Fig.7). CREB4
is an ER-resident transmembrane transcription
factor that is structurally similar to OASIS andcontains a bZIP domain in its cytoplasmic
region. Because OASIS also possesses a bZIP
domain, CREB4 may form a heterodimer withOASIS that promotes the expression of target
genes required for the differentiation of goblet
cells. Previous characterization of Creb4
knockout mice revealed anomalies inspermatogenesis (47), but a detailed analysis of
the gut has not conducted. Because CREB4
could be a key molecule for the maturation ofgoblet cells, further examination of Creb4-/mice may enable further understanding of theGoblet cells in the large intestine produce
and secrete abundant mucus, which provides aprotective barrier against bacteria and otherharmful stimuli. Defects in this barrier
contribute to chronic diseases, including colitis
and cancer. Muc2-/- mice have been observed
to develop spontaneous colitis (48). Similarly,
intestinal trefoil factor-deficient mice exhibit
increased susceptibility to dextran sodium
sulfate-induced colitis (49). Therefore,
impairment of goblet cell maturation may belinked to inflammation of the intestinal mucosa.
Because mature goblet cells in Oasis-/- miceare drastically decreased, it is possible that themice are also sensitive to colitis. Targeted
deletion of the ER stress response transcription
factor, XBP1, and the ER stress sensor, IRE1β,causes sensitivity to experimental colitis(50-52). However, the mechanisms of which
are sensitive to colitis are not consistent withthe case of mice deleted goblet celldifferentiation-related genes such as Muc2 orIntestinal trefoil factor. Loss of Xbp1 or Ire1β
induces severe ER stress in goblet cells, andleads to apoptosis and chronic inflammation in
the large intestine. We observed abnormallyexpanded rough ER in the goblet cells of
Oasis-/- mice, suggesting that the function of
the ER in Oasis-/- goblet cells may be impaired
due to sensitivity to ER stress. Thus, OASIS
may play roles not only in the differentiation ofgoblet cells, but also in protecting these cells
from ER stress. Indeed, overexpression of
OASIS was reported to protect against ER
stress-induced cell death (17). Although further
examination into the relationship between
OASIS function and the onset of colitis isrequired, it is possible that OASIS is a novel
drug target for inflammatory bowel diseases
such as ulcerous colitis.

8

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FOOTNOTES
We thank S. Nakagawa for technical support. This work was partly supported by grants from the Japan
Society for the Promotion of Science KAKENHI (#22020030, #22800049), Sumitomo Foundation,
Mochida Memorial Foundation for Medical and Pharmaceutical Research, Astellas Foundation for
Research on Metabolic Disorders, Takeda Science Foundation, The Pharmacological Research
Foundation Tokyo, Daiichi-Sankyo Foundation of Life Science, and The Naito Foundation.
The abbreviations used are: bZIP, basic leucine zipper; ER, endoplasmic reticulum; UPR, unfolded

10

protein response; OASIS, old astrocyte specifically induced substance; BBF-2, box B-binding factor-2;
BBF2H7, BBF2 human homolog on chromosome 7; CREB, cyclic AMP-response element-binding
protein; CREB4, cyclic AMP-response element-binding protein 4; BiP, immunoglobulin heavy
chain-binding protein; ERAD, ER-associated degradation; Col1, type I collagen; SPDEF, SAM-pointed
domain-containing ETS-like factor; Atoh1, atonal homolog 1; Hes1, hairy and enhancer of split 1; Dll1,
delta-like 1; Gfi1, growth factor independent 1; Muc2, muchin 2; Agr2, anterior gradient 2; Retnlb,
restin like beta; Tff3, trefoil factor 3; XBP1, X-box binding protein 1; IRE1, inositol requiring 1;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase

FIGURE LEGENDS
Fig. 1. The expression of Oasis mRNA in the large intestine. A, RT-PCR analysis of Oasis mRNA invarious tissues from 3-week old mice. Oasis mRNA is highly expressed in the large intestine. B, The
expression of Oasis mRNA in each region of the large intestine. Right; quantitative analysis of Oasis
mRNA. C, Hematoxylin-Eosin staining (HE) in the large intestine. Many goblet cells withmucus-containing vacuoles are observed in the crypt. D and E, in situ hybridization analysis of Oasis
mRNA (D) and Gapdh mRNA (E) in the large intestine. Oasis mRNA is expressed in the lower portionof the crypt.

Fig. 2. Anomalies of goblet cells in the large intestine of Oasis-/- mice. A, HE staining; B, PAS
staining. The numbers of goblet cells with vacuoles are severely decreased in Oasis-/- mice. C and D,
PAS-Hematoxylin counter staining of WT and Oasis-/- large intestine. C, proximal large intestine; D,
distal large intestine. E, The percentages of PAS-positive goblet cells in the crypt of the large intestine in
WT and Oasis-/- mice. Data are mean ± s.d., n=5, ***P < 0.001, unpaired Student’s t-test.

Fig. 3. Electron microscopy of goblet cells in the large intestine. A and B, images of goblet cells at
the crypt base in the large intestine. C and D, high magnification of the rough ER in panels A and B.
Note that rough ER in Oasis-/- goblet cells displays aberrant expansion. E and F, goblet cells in theupper portion of the crypt. The numbers of mucus vesicles are decreased and the membrane of somevesicles are often fused in goblet cells of Oasis-/- large intestine. Scale bars, 4μm (A, B, E, and F),
1.6 μm (C and D)

Fig. 4. The expression of goblet cell markers in the large intestine. A, RT-PCR of genes involved inprimary differentiation from intestinal stem cells and an early goblet cell marker, Tff3. Note that theexpression of Tff3 is significantly elevated in Oasis-/- mice. B, RT-PCR of mature goblet cell markers.
C, RT-PCR analysis of Spdef and Creb4. Data are mean ± s.d., n=3, *P < 0.05, **P < 0.01, unpaired
Student’s t-test. D-F, in situ hybridization of Tff3 (D), Muc2 (E), and Spdef (F) in the large intestine.

11

Fig. 5. Gene expression and OASIS activation during goblet cell maturation. A, PAS staining of
LS174T cells 24 hrs after treatment with sodium butyrate. LS174T cells are completely differentiated tomature goblet cells containing PAS-positive mucus 24 hrs following treatment. B, RT-PCR of maturegoblet cell markers in LS174T cells treated with sodium butyrate. The expression levels of maturegoblet cell markers are increased after treatment with sodium butyrate. C, Western blot analysis of
OASIS in LS174T cells treated with sodium butyrate (left). Quantification of the amounts of OASIS
N-terminus (right). D, RT-PCR of ER stress markers during LS174T cell differentiation. E, RT-PCR of
Spdef and Creb4 mRNAs in LS174T cells treated with sodium butyrate. Data are mean ± s.d., n=5.

Fig. 6. OASIS is required for the terminal differentiation of goblet cells. A, RT-PCR of Oasis
mRNA in LS174T cells 12 hrs after transfection with Oasis siRNA. The expression of Oasis mRNA issignificantly suppressed by the siRNA. B, PAS staining of LS174T cells 24 hrs after treatment withsodium butyrate. Oasis knockdown results in inhibition of goblet cell maturation. C-E, RT-PCR of
mature goblet cell markers (C), Spdef and Creb4 (D), and ER stress markers (E) in LS174T cells treatedwith sodium butyrate following transfection with Oasis siRNA. Data are mean ± s.d., n=5. *P < 0.05,**P < 0.01, unpaired Student’s t-test.

Fig.7. Putative roles of OASIS in the differentiation of goblet cells in the large intestine.
SUPPLEMENTAL TABLE LEDEND
Table 1. RT-PCR was performed using each specific primer set.
Table2. Digoxigenin-labeled probes for each gene.

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Figure 5A+ Sodium butyrate

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intestinal stem cell

Secretory progenitor cell

Starting productionof abundant proteins
Early goblet cell
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SPDEFTerminal maturation(CREB4 and other factors)

Mature goblet cell19