Absence of keratin 8 confers a paradoxical
microflora-dependent resistance to apoptosis
in the colon
Aida Habteziona,b,1,2, Diana M. Toivolac,1, M. Nadeem Asgharc, Greg S. Kronmald, Jacqueline D. Brookse,
Eugene C. Butcherb,f, and M. Bishr Omaryg,h
Departments ofgMolecular and Integrative Physiology andhMedicine, University of Michigan Medical School, Ann Arbor, MI 48109; Departments of
aMedicine andfPathology, Palo Alto VA Medical Center, Palo Alto, CA 94304;bStanford University School of Medicine Digestive Disease Center, Stanford, CA
94305;cDepartment of Biosciences, Åbo Akademi University, FIN-20520, Turku, Finland;dApplied Biosystems Inc., Foster City, CA 94404; and
Inc., Redwood City, CA 94063
Edited by Sven Pettersson, Karolinska Institutet, Stockholm, Sweden, and accepted by the Editorial Board December 16, 2010 (received for review July
Keratin 8 (K8) is a major intermediate filament protein present in
enterocytes and serves an antiapoptotic function in hepatocytes.
K8-null mice develop colonic hyperplasia and colitis that are
reversed after antibiotic treatment. To investigate the pathways
that underlie the mechanism of colonocyte hyperplasia and the
normalization of the colonic phenotype in response to antibiotics,
we performed genome-wide microarray analysis. Functional anno-
tation of genes that are differentially regulated in K8−/−and K8+/+
isolated colon crypts (colonocytes) identified apoptosis as a major
altered pathway. Exposure of K8−/−colonocytes or colon organ
(“organoid”) cultures, but not K8−/−small intestine organoid cul-
tures, to apoptotic stimuli showed, surprisingly, that they are re-
sistant to apoptosis compared with their wild-type counterparts.
This resistance is not related to inflammation per se because T-cell
receptor α-null (TCR-α−/−) and wild-type colon cultures respond
similarly upon induction of apoptosis. Following antibiotic treat-
ment, K8−/−colonocytes and organ cultures become less resistant
to apoptosis and respond similarly to the wild-type colonocytes.
Antibiotics also normalize most differentially up-regulated genes,
including survivin and β4-integrin. Treatment of K8−/−mice with
anti–β4-integrinantibodyup-regulated survivin,and induced phos-
phorylation of focal adhesion kinase with decreased activation of
caspases. Therefore, unlike the proapoptotic effect of K8 mutation
or absence in hepatocytes, lack of K8 confers resistance to colono-
cyte apoptosis in a microflora-dependent manner.
make up the intermediate filament (IF) cytoskeleton of epithelial
cells (1–3). In adult hepatocytes, the IF network consists of simple
of K7, K8, K18, K19, and K20, with K8 being the major type II
keratin (2, 4, 5). Absence or mutation of K8 or K18 renders hep-
atocytes markedly susceptible to apoptosis, and in humans and
mice K8 and K18 mutations predispose their carriers to acute and
chronic end-stage liver disease and liver disease progression (5–8).
colonic hyperplasia and chronic spontaneous colitis (9, 10) that is
amenable to early treatment with broad-spectrum antibiotics (11).
Although hepatocytes in K8−/−and K18−/−mice are highly
sensitive to apoptotic stimuli (5, 12, 13), K18−/−intestine appears
normal (14), probably reflecting the functional redundancy of ad-
ditional type I keratins in the intestine (4, 15). In humans, the as-
sociation of K8 variants with inflammatory bowel disease (IBD) is
unclear (16, 17). The differences between the liver- and intestine-
human disease highlight the potential importance of the micro-
environment and cell-specific modifiers.
In contrast to the findings in K8−/−hepatocytes, we show in
this study that K8−/−colonocytes, but not K8−/−small intestine
enterocytes or T-cell receptor α-null (TCRα−/−) colonocytes, are
eratins exist as obligate noncovalent heteropolymers of type I
(K9–K28) and type II (K1–K8 and K71–K80) proteins and
in K8−/−mice by antibiotic treatment. In addition, we show that
K8−/−colonocytes up-regulate survivin and β4-integrin, with the
keratin IF network links to β4-integrin at the site of hemi-
desmosomes via interaction with the cytoskeletal linker protein
plectin and BP180 (20, 21). Furthermore, we provide a mecha-
nism for the altered susceptibility to apoptosis, because in vivo
treatment of K8−/−mice with anti–β4-integrin antibody further
up-regulates survivin, leads to the activation of down-stream
phosphorylation of focal adhesion kinase (FAK), and decreases
caspase activation. The resistance to apoptosis observed at the tip
of the colonic crypt coupled with colonocyte proliferation along
most of the crypt likely contribute to the observed colonic
hyperproliferation in K8−/−mice.
Expression Profiling in K8−/−and K8+/+Colonocytes Shows Apoptosis
Is a Prominent Keratin-Regulated Biological Pathway. Tounderstand
better the effect of K8 absence on the colonic hyperplasia and
colitis phenotype and the normalization of this phenotype fol-
lowing antibiotic treatment (11), we sought to identify colonocyte
genes that are differentially expressed in response to the absence
of K8 or to suppression of luminal bacteria. Microarray analysis
revealed that several hundred genes were differentially regulated
The 20 genes most highly up-regulated in the K8−/−colon crypts
are listed in Table S1. Antibiotic pretreatment of the animals led
to near-complete normalization of the differentially altered genes
in the K8−/−colon (Fig. 1B). The antibiotics used herein effec-
tively suppressed the presence of luminal bacteria as determined
by stool bacterial DNA content (Table S2). Using real-time PCR,
we validated several of the genes that manifested marked up- or
down-regulation in the microarray findings (Table S3). Analysis
using the Jubilant PathArt database package Physiology software
revealed that apoptosis was the most differentially regulated bi-
ological pathway, with 46 of 160 apoptosis pathway genes showing
significant differential expression (P = 3 × 10−5) in K8−/−and
K8+/+colonocytes (Table S4). The software identified growth
Author contributions: M.B.O. designed research; A.H., D.M.T., M.N.A., and M.B.O. per-
formed research; G.S.K., J.D.B., E.C.B., and M.B.O. contributed new reagents/analytic
tools; A.H., D.M.T., and M.B.O. analyzed data; and A.H., D.M.T., and M.B.O. wrote the
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. S.P. is a guest editor invited by the Editorial
1A.H. and D.M.T. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| January 25, 2011
| vol. 108
| no. 4
of 135 genes differentially expressed in K8−/−and K8+/+colo-
nocytes (P = 1 × 10−4).
K8−/−Colonocytes Overexpress Survivin and, Unlike K8−/−Cells from
the Small Intestine and K8−/−Colonocytes from Antibiotic-Treated
Mice, Are Resistant to Apoptosis. In the human small intestine, it
is estimated that 1010cells are shed per day; despite the large
number of cells being shed, the epithelial barrier is maintained
(22), enterocytes undergo a spontaneous form of apoptosis,
termed “anoikis,” upon detachment from the cellular matrix (18).
Because of the established predisposition of K8−/−(and K8, K18
mutant) hepatocytes and livers to mechanical and nonmechanical
injury and to apoptosis (23–28), we compared the apoptotic re-
sponse of K8−/−and K8+/+small intestine and colons. K8−/−co-
lonic crypts (here called “colonocytes”) were isolated in parallel,
apoptosis. Surprisingly, and unlike K8−/−hepatocytes, the K8−/−
colonocytes are resistant to apoptosis based on the relative de-
crease in cleavage of caspase-3, -7, and -9 (Fig. 2A). Activation of
apoptosis also was associated with K8 S80 phosphorylation in
K8+/+colonocytes (Fig. 2A), and ablation of S80A in hepatocytes
by an S80A mutation leads to marked predisposition to apoptosis
using whole-organ fragments (here called “organoids”) of the
colon (Fig. 2B), but not of the small intestine (Fig. 2C), that were
kept in culture for 1 h. Notably, small intestine organoids from
K8−/−mice appear to be more predisposed to apoptosis as com-
pared to K8+/+small intestine based on the enhanced cleavage of
caspase-7 (Fig. 2C). Consistent with susceptibility to apoptosis
after 1 h culture, K18 fragmentation (K18 Asp237) (30) was
detected in both colon and small intestine organoid K8+/+cul-
tures (Fig. 2 B and C). To ensure that caspase activation involved
colonocytes rather than lamina propria or other nonepithelial
cells, we analyzed cleaved caspase-7 in frozen colon pieces from
the organ cultures by immunofluorescence staining (Fig. 2D). A
significant increase in apoptotic cells was seen at the luminal or
surface end of the K8+/+colonic crypts, compared with K8−/−
colonic crypts, andthecells were identifiedas colonocytes by their
coexpression of K19 (Fig. 2D a and b).
and Fas antibody to examine potential differential sensitivity to
apoptosis of K8−/−and K8+/+colonocytes. Similar to findings
noted during colonocyte isolation (Fig. 2), K8−/−colons are more
resistant than K8+/+colons to the induction of apoptosis by both
staurosporine and Fas. This finding was confirmed biochemically
by the limited formation of cleaved caspase-7 (Fig. 3A) and by
immunofluorescence staining (Fig. 3B). In addition, the resistance
of K8−/−colonocytes to apoptosis, compared with K8+/+colono-
consistent with the “normalization” of the differentially regulated
genes in K8−/−and K8+/+colonocytes in antibiotic-treated mice,
K8−/−colonocyte resistance to apoptosis was reversed with anti-
biotic pretreatment (Fig. 3 C and D).
se or inflammation triggered by microbes contributes to the ob-
served alteration in apoptosis, we examined the differences in
colonocyte apoptosis in another chronic colitis model, namely the
TCRα−/−model of chronic colitis (31). Both TCRα−/−and K8−/−
creased numbers of differentially regulated genes after antibiotic treatment.
(A) Total RNA from freshly isolated K8−/−and K8+/+colonocytes was used for
microarray analysis. The graph depicts results from GeneSpring analysis. Up-
K8+/+compared with K8−/−colonocytes. (B) An analysis to that similar in A
exceptthat theRNA was isolated from colonocytes of antibiotic-treated K8−/−
and K8+/+mice. Note the marked decrease in differential expression between
K8−/−and K8+/+colonocyte genes following antibiotic treatment. Two hun-
dred eighty-eight genes were threefold up-regulated, and 460 genes were
threefold down-regulated in K8+/+compared with K8−/−colonocytes, but af-
regulated and down-regulated, respectively.
Gene expression profiles of K8−/−and K8+/+colonocytes show de-
small intestine organ cultures from K8−/−mice
are less susceptible to apoptosis than parallel
cultures from K8+/+mice. (A) K8+/+and K8−/−
colonocytes were cultured in medium for 1 h.
Cell homogenates then were analyzed by im-
munoblotting using antibodies to cleaved cas-
pases (cCasp) or total caspases (Casp), K8 pS80,
K8, and tubulin. (B) Colon and (C) small intestine
organoids were cultured and then analyzed as
in A. (D) Frozen sections from K8+/+(a and b)
and K8−/−(c and d) 1-h colon primary organ
cultures were triple stained for anti–cleaved
caspase-7 red), anti-K19 (green), and nuclei
(blue). Merged images are shown in b and d.
Arrows point to apoptotic cells at the luminal (L)
tips of K8+/+crypts. Insets show magnified views.
(Scale bar in c: 50 μm.)
Primary colonocytes and colon but not
| www.pnas.org/cgi/doi/10.1073/pnas.1010833108Habtezion et al.
mice manifest a T-helper type 2 (Th2) type of colitis (11, 31).
Unlike the situation in K8−/−colon (Fig. 3 A and B), TCRα−/−
colonocytes were not resistant to apoptosis compared with their
wild-type counterparts (Fig. S2). In agreement with the histologi-
cally evident K8−/−colonic hyperproliferation, Ki67 staining was
increased in K8−/−compared with K8+/+colonocytes (Fig. 4A).
Quantification showed a significant increase in the number of
Ki67+cells present in K8−/−colonic crypts, even when normalized
to crypt length (0.12 μm−1vs. 0.05 μm−1, P < 0.001). As expected,
most of the Ki67+cells are present at the bottom half of the crypt,
but they can be found in the lower three-fourths of the crypt.
Consistent with the findings from Ki67 staining, immune blotting
(9) shows increased numbers of proliferating cell nuclear antigen
(PCNA)-positive cells in K8−/−compared with K8+/+colons.
Given the observed decreased susceptibility of K8−/−colono-
cytes to apoptosis, we used the microarray data to survey the
antiapoptosis genes that were differentially regulated. Among the
differentially regulated genes, survivin levels increased in K8−/−
colonocytes by 3.6-fold with microarray analysis and by 5.7-fold
with quantitative PCR (qPCR) analysis (Table S3). Survivin is
a member of the inhibitor of apoptosis family with a dual role
based on its localization: nuclear survivin is involved in chromo-
somal complex package during cell division, and cytoplasmic
survivin plays an important role in inhibiting apoptosis (32, 33).
Immunofluorescence staining showed similar levels of survivin
protein in K8+/+and K8−/−nuclei, but survivin was particularly
up-regulated in the cytoplasm of the K8−/−colonocytes (Fig. 4B c
and d), suggesting that survivin plays an important role in con-
ferring resistance to apoptosis.
K8−/−Colonocytes Have Altered β4-Integrin Expression. Studies in
keratinocytes showed that cytoplasmic survivin can be down-
regulated via blockade of β1-integrin, thereby leading to increased
anoikis (34). This finding led us to compare integrin expression in
K8−/−and K8+/+colons. K8−/−colonocytes, particularly in the
proximal colon [where inflammation is more prominent than in
the distal colon (Fig. S3)], express higher levels of β4-integrin
(Figs. 4C and 5A) but not β1-integrin (Fig. 5 B and C) compared
with K8+/+colons. β4-integrin is known to be distributed uni-
formly along the crypt–villus axis (35), and the increase in β4-
integrin staining is seen in both the base and tip of the K8−/−co-
lonic crypts (Fig. 4C).
In Vivo Anti–β4-Integrin Antibody Administration Leads to Further
Increase in Survivin, FAK Phosphorylation, and Inhibition of Apoptosis.
It is known that integrin-mediated cell-to-matrix contact leads to
signals, probably through Akt (18). Based on this information, we
examined whether the increased expression of β4-integrin in K8−/−
colonocytes is functional by measuring consequent FAK phos-
phorylation in vivo using an activating anti–β4-integrin antibody.
The activating nature of the anti–β4-integrin antibody we used was
demonstrated by its ability to induce Akt phosphorylation using
two mouse mammary cell lines, JC and EMT6 (results for JC are
shown in Fig. 6A), as has been shown for other clones of anti–β4-
resistance to apoptosis. Colon organ cultures from K8 mice
not treated (A and B) or treated with antibiotic (C and D)
were maintained in the presence or absence of staur-
osporine (STS) or Fas for 1 h. Livers from FVB/n mice treated
with Fas antibody (i.p. administration) for 4 h or not trea-
ted were included as positive and negative controls, re-
spectively, for formation of cleaved caspase-7 (cCasp7). The
organoid cultures then were processed for immunoblotting
(A and C) or antibody staining (B and D). Antibody staining
was done for anti–cleaved caspase-7 (red) and nuclei (blue).
Note the resistance to apoptosis in colons from K8−/−mice
not treated with antibiotic (A and images d–f in B) and the
reversal of this resistance to apoptosis in colons from K8−/−
mice treated with antibiotics (C and images d–f in D). Casp7,
caspase 7; L, lumen.
Antibiotic treatment of mice reverses the K8−/−
K8−/−colon. (A) Paraffin-embedded colon sections from K8+/+and K8−/−mice
stained with Ki67 demonstrate increased colonocyte proliferation in K8−/−
compared with K8+/+mice. (Scale bar: 10 μm.) An average of 70–100 crypts
per colon was analyzed. Quantification showed a significant increase in the
number of Ki67+cells in K8−/−colonic crypts, normalized to crypt length
(0.12 μm−1versus 0.05 μm−1). P < 0.001, when comparing K8+/+versus K8−/−;
n = 3 pairs. (B) Frozen colon sections from 3-mo-old K8+/+(a and b) and K8−/−
(c and d) mice were triple stained for survivin (red), K19 (green), and nuclei
(blue). Merged images for the indicated double or triple staining are shown.
Increased survivin, especially in the cytoplasm, was seen in K8−/−colons
(arrows in c and d). (C) Frozen colon sections (crypt tips, Left; bases, Right)
from K8+/+(a and b) and K8−/−(c and d) mice were double stained for β4-
integrin (green), and nuclei (blue). Note increased β4-integrin staining
(arrows) in the basolateral compartment of K8−/−epithelial cells (c and d).
(Scale bar: 10 μm.) L, lumen.
Altered proliferation and expression of survivin and β4-integrin in
Habtezion et al.PNAS
| January 25, 2011
| vol. 108
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integrin antibody using human breast and other cell lines (36, 37).
We then tested the effect of β4-integrin antibody treatment on
FAK phosphorylation and down-regulation of caspase activation
in vivo. Notably, treatment of K8−/−mice with anti–β4-integrin
antibody led to an increase in survivin mRNA (Fig. 6B) and pro-
tein (Fig. 6C).
The specificity of the anti–β4-integrin antibody is demonstrated
by the fact that FITC anti-rat IgG stained β4-integrin in colono-
cytes of K8−/−mice treated with anti–β4-integrin antibody (Fig.
6C b and d) but did not stain colonocytes from mice treated with
the isotype control antibody (Fig. 6C a and c). Furthermore,
treatment with anti–β4-integrin antibody led to a robust increase
in FAK pY397 in K8−/−colons (Fig. 6D). Although phosphory-
lation of Akt is increased in K8−/−compared with K8+/+colons,
immune blotting did not show significant differences in phospho-
Akt between K8−/−mice treated with isotype control and K8−/−
mice treated with anti–β4-integrin antibody (Fig. 6D). However,
binding of β4-integrin leads not only to an increase in survivin
expression (Fig. 6 B and C) but also to inhibition of apoptosis, as
shown by the decrease in cleavage of caspase-7 (Fig. 6D). In ad-
dition, we assessed the activation of phosphatase and tensin ho-
molog deleted on chromosome 10 (PTEN), because PTEN has
induction during apoptosis that is in part Akt dependent (38). We
do not observe any evidence of PTEN activation (Fig. 6D). Col-
lectively, these findings indicate that the up-regulation of β4-
integrin in the K8−/−colon is likely to contribute to the observed
inhibition of apoptosis via FAK activation.
K8−/−and K18−/−(or keratin mutant) hepatocytes have similarly
increased susceptibility to injury and apoptosis (5, 12, 13) because
of the obligate heteropolymeric nature of K8/K18 and because,
under basal conditions, K8 and K18 are the only keratins in adult
hepatocytes (5). In the colon, K8 is the major type II keratin in
mice (4) and humans (17) and is the major keratin that manifests
a phenotype when absent (9, 10, 39) probably because of the
functional redundancy of the type I keratins in the colon and the
limited expression of K7 compared with K8 (5). Of the remaining
keratins (K7, K18, K19, K20) concurrently expressed in the colon,
the absence of K18 (14) or K19 (40, 41) or the overexpression of
(to our knowledge, K7−/−mice have not been reported). In the
liver, K8 is antiapoptotic; in the colon, as shown here, its absence
conditions that include the presence of resident microflora. The
increased susceptibility of keratin-lacking or keratin-mutant hep-
atocytes to apoptosis, compared with their wild-type counterparts,
is context dependent, in that differences are unmasked when ap-
optosis is induced by Fas but not by TNF (26, 42). Similar context-
dependent bacteria-modulated effects alsomayoccurinthecolon,
but this possibility remains to be investigated. It also is plausible
that absence of normal keratins induces resistance to apoptosis as
part of a normal stress response to luminal bacteria.
The mechanism by which microflora provide an antiapoptotic
effect in the absence of colonocyte keratins is likely to be multi-
factorial. A relevant mechanism demonstrated herein is the en-
expression. (A) Lysates from proximal (PC) and distal colons
(DC) shown from three independent age- and sex-matched
pairs of K8+/+and K8−/−mice were blotted with antibodies
to β4-integrin and actin. Note the increase in β4-integrin
(arrow) in proximal colons of K8−/−mice. (B) Lysates from
proximal (PC) and distal colons (DC) from K8+/+and K8−/−
mice were blotted with antibodies to β1-integrin and actin. The blot from a representative pair of K8 mice is shown. (C) Frozen colon sections from K8+/+(a)
and K8−/−(b) mice were double stained for β1-integrin (red) and nuclei (blue). (Scale bar: 10 μm.) L, lumen.
K8−/−colons have altered β4- but not β1-integrin
anti–β4-integrin antibody increases the expres-
sion of survivin and activation of FAK in concert
mouse mammary cell line treated with control or
anti–β4-integrin antibody were analyzed by im-
munoblotting using antibodies to Akt, phospho-
isotype control antibody (white bars) or anti–β4-
as mean relative survivin expression ± SEM (n =
3). *P < 0.05 when comparing control versus
anti–β4-integrin antibody treatment. (C) Frozen
colon sections from K8−/−mice treated with iso-
type control (a and c) or anti–β4-integrin anti-
body (b and d) were triple stained with anti-
survivin (red), anti-rat IgG (green), and nuclei
(blue). Higher-magnification images are shown
incand d. (Scale bars: 50μm ina andb; 10μm inc
and d.) (D) Lysates from K8+/+and K8−/−colons of
mice treated with control antibody or anti–β4-
integrin antibody were analyzed by blotting us-
ing antibodies to FAK, Akt, cleaved caspase-7
(cCasp7), caspase-7 (Casp7), tubulin, and phos-
pho-FAK/Akt/PTEN. (E) Schematic model sum-
marizing the proposed effect of K8 absence on
colonocyte resistance to apoptosis through in-
creased integrin signaling and increased cyto-
to the observed hyperproliferative epithelium in K8−/−colon.
In K8−/−colon, in vivo treatment with
| www.pnas.org/cgi/doi/10.1073/pnas.1010833108 Habtezion et al.
hanced integrin signaling in the K8−/−colon, which activates FAK
and is mimicked and enhanced by administration of β4-integrin
antibody. We also link K8 absence to increased levels of survivin,
which is known to be involved, together with an activated FAK, in
render keratin absence antiapoptotic may be related to quantita-
are required for the intestinal homeostasis and protection of in-
testinal epithelial cells from injury (44, 45). For example, mis-
targeting oralteredexpressionof apical orbasolateral receptorsin
K8-null enterocytes, as seen in the small intestine (39), colon (9),
or more TLRs. A survey of the microarray results of several TLRs
showed that TLR9 mRNA increased 2.5-fold, (particularly in the
proximal colon; Fig. S4), a finding that was confirmed by immu-
nohistochemistry and immunoblotting. Although the exact mech-
K8 and up-regulation of β4-integrin expression in the colon. This
transgenic mice overexpressing human K8 (Fig. S5).
The importance of the microflora also is highlighted by the
difference in resistance to apoptosis observed in the K8−/−colon
and small intestine (jejunum). The human colon harbors 1013–
1014bacteria, whereas the proximal small intestine is estimated
to have 103–104bacteria (46). Factors other than microflora also
may be involved in the differential resistance to apoptosis, be-
cause the small intestine and colon epithelium differ in many
respects, ranging from morphologic to functional states. It is
unlikely that differences in the composition of the keratin net-
work in K8−/−colon and small intestine account for the differ-
ence in the two sites’ resistance to apoptosis, because previous
studies showed similar alterations at the two sites in the
remaining keratins (K7, K18, K19, and K20) (10, 39).
Colonic inflammation parallels the presence of luminal micro-
flora. It is possible that the observed resistance of K8−/−colono-
cytes to apoptosis may be secondary to, or influenced by,
inflammation. Inflammation is difficult to assess in the absence of
luminal microflora because inflammation and microflora go hand
in hand. Despite the different genetic or chemical mechanisms
used to generate the colitis models, none of the current models
develop colonic inflammation in a germ-free environment (47).
another immune-based model of chronic colitis, TCRα−/−mice.
We did not observe such resistance to apoptosis in the TCRα−/−
model of colitis, which by definition harbors an inflamed epithe-
lium (31). However, our studies do not control for possible con-
tributions arising from strain differences or differences in
The findings herein highlight the importance of differences (such
as microflora) in the microenvironments of hepatocytes and
colonocytes; these differences lead to a paradoxical effect in the
colon. If this effect also applies to hepatocytes, it might serve (e.g.,
through microflora-mimetic stimulation) to protect them from
hepatotoxic injury. Taken together, these findings demonstrate
that the absence of K8, coupled with the presence of microflora,
renders colonocytes resistant to apoptosis. This resistance appears
that can be linked to an increase in survivin and subsequent de-
creased activation of caspases (Fig. 6E).
The colonic hyperplasia that is observed in K8−/−mice (9, 10)
probably results from both decreased apoptosis (Figs. 2 and 3 and
Fig. S1) and increased proliferation (Fig. 4). We used two forms
of stress to induce apoptosis: (i) colonocyte (colonic crypts) or
whole-colon tissue isolation (organoids) followed by ex vivo cul-
ture, and (ii) application of known apoptosis-inducing agents ex
vivo. The former method is similar to the classically described
anoikis (a spontaneous form of apoptosis resulting from de-
tachment from the cellular matrix), which occurs upon suppres-
sion of β4-integrin signaling (18). Therefore, the endogenous up-
regulation of β4-integrin signaling in situ in K8−/−colon (Fig. 6)
provides one likely mechanism for the observed protection from
apoptosis. This protection occurs at the crypt surface, whereas the
increase in proliferation occurs below this level, mostly involving
the crypt from its base to ≈75% of its upward length (based on
the Ki67 staining; Fig. 4A). These findings suggest that both re-
sistance to apoptosis and the increase in proliferation probably
account for the colonic hyperplasia. The microarray data and
pathway analysis also indicated that the growth and differentia-
tion pathway is the second most significantly altered pathway
(Table S4), providing further indirect evidence for the dual, and
probably regionally distinct, enhanced antiapoptosis and hyper-
proliferation signals as a consequence of the combined absence of
K8 and presence of microflora. Other contributions to enhanced
cell proliferation in the K8−/−colon probably are related to the
mistargeting and alteration of the stability of membrane proteins
such as transporters that can modulate intracellular pH and en-
hance cell proliferation (9).
Materials and Methods
Mice and in Vivo Experiments. K8−/−mice(JacksonLaboratory); andtheirwild-
type littermates, in an FVB/N background, were generated by interbreeding
K8+/−mice as described (11). Age- and sex-matched mice (3–4 mo old) were
studied. For antibiotic treatment, K8+/+and K8−/−mice were given vanco-
mycin and imipenem in their drinking water (50 mg/kg body weight/d for
8 wk) starting at postnatal d 18 or 19 (11). A group of K8+/+and K8−/−mice
received 200 μg anti–β4–integrin antibody (clone 346–11A) or control isotype
antibody (rat IgG2a; BD Biosciences) i.p. every day for 3 d, and colons were
harvested on d 4. In separate experiments, FVB/N mice were fasted overnight,
Biosciences) as described (42). After 4 h, the livers were harvested, and their
homogenates were used as positive controls for caspase cleavage and apo-
ptosis. TCRα+/+and TCRα−/−mice on a C57BL/6 background were obtained
fromthe JacksonLaboratory. Mice overexpressinghuman K8were generated
and maintained as described previously (48). All animals were treated
according to National Institutes of Health guidelines and an approved animal
Colonocyte Isolation and Intestine and Other Cell Culture. Colonocytes were
isolated as described (9). Isolated colonocytes were used for microarray
analysis (see below) or were cultured in Dulbecco’s modified Eagle medium
supplemented with 4.5 g/L glucose, 25 mM Hepes, 5 ng/mL recombinant
human epidermal growth factor, 0.2 IU/mL insulin, 5% FBS, 50 μg/mL peni-
cillin, and 50 μg/mL streptomycin at 37 °C for 1 h. For organoid whole-colon
or small intestine cultures, organs were opened longitudinally and were
rinsed gently and quickly in incubation medium (RPMI-1640 plus 10% heat-
inactivated FCS and antibiotics). The intestines then were cut into 5- to 7-mm
pieces and incubated in the culture medium described above for 1 h at 37 °C,
5% CO2/95% O2. The incubations were carried out in the presence or ab-
sence of the apoptosis-inducing agents staurosporine (4 μM) (Cayman
Chemical) and Fas antibody (500 ng/mL).
Activity of the anti–β4-integrin antibody or isotype control was tested in
cultured mouse mammary cell lines (JC and EMT6; American Type Culture
Collection). The cells were serum starved overnight, trypsinized from the
dishes, and washed with PBS. The cells (5 × 106/mL) then were cultured at
37 °C for 6–8 h in the presence of anti–β4-integrin or isotype control anti-
body (6 μg/mL).
Real-Time PCR. Total colon RNA was isolated using an RNeasy midi kit and
converted into cDNA using a SuperScript II reverse transcriptase kit as rec-
ommended by the supplier (Invitrogen). qPCR was performed with an ABI
Prism 7900 Sequence Detection System as described (49). Target genes (Table
S3) were amplified using specific primers and SYBR Green PCR Master Mix.
Gene expression levels were normalized to the housekeeping gene GAPDH.
Bacterial DNA was extracted from freshly collected stools using the QIAamp
DNA stool kit (Qiagen). Escherichia coli plasmid with a known amount of
DNA (a gift from David Relman, Stanford University, Stanford, CA) served as
a positive control, and stool DNA qPCR was performed as described (50).
Histology. Freshly harvested colons or organ-culture colon pieces from K8+/+
and K8−/−mice were embedded in optimum cutting temperature (OCT)
compound and frozen at –80 °C. Frozen 6-μm tissue sections were fixed in
acetone and blocked with PBS containing 2% BSA and 2% goat serum (11,
51). The tissue sections were incubated with antibodies directed to cleaved
caspase-7 (Cell Signaling Technology), K19 (Troma III; Developmental Studies
Hybridoma Bank), survivin (Abcam), β4-integrin (BD Biosciences), or β1-
Habtezion et al.PNAS
| January 25, 2011
| vol. 108
| no. 4
integrin (R&D Systems) followed by washing and then incubation with the Download full-text
secondary antibodies, Texas Red or FITC goat anti-rabbit or anti-rat IgG
(Jackson ImmunoResearch). Nuclei were stained after RNase treatment (51)
using either Toto-3 or DAPI. Images were captured using a Zeiss LSM510
confocal microscope. Ki67 and PCNA staining of paraffin-embedded colon
sections was performed by Histo-Tec Laboratory.
TUNEL Staining. Organ-culture colon pieces from K8+/+and K8−/−mice were
embedded in OCT compound and frozen at –80 °C. Frozen tissue sections
(6 μm) were stained using ApopTag Red In Situ Apoptosis Detection Kit
Immunoblotting. Total liver and intestine homogenates were prepared in
sample buffer containing 3% SDS. Except where indicated, we used total
colons, because colons are shorter in K8−/−mice than in K8+/+mice. Isolated
colonocyte lysates were prepared in the same buffer by shearing with a 1-mL
syringe mounted with a 21-G needle. Similarly, cell lysates were prepared
from cultured JC and EMT6 cell lines by scraping the cells from the wells
and shearing. Proteins were separated using SDS/PAGE and then were
transferred to membranes. Membranes were blotted with the primary anti-
bodies: anti-caspases, anti-cleaved caspases, anti-FAK, anti-pPTEN, anti–pAkt,
anti-Akt (Cell Signaling Technology); anti-pFAK (Upstate Biotechnology);
anti-K8 (Troma I); anti-K8 pS79 (LJ4) (51); anti–β4-integrin (BD Biosciences);
anti–β1-integrin (R&D Systems); anti-tubulin, or anti-actin (NeoMarkers).
Proteins were visualizedusing Western LightningTMChemiluminescence Plus
Statistical Analysis. Data are expressed as means ± SEM from at least three
independent experiments. Significance of differences was determined using
the two-tailed Student’s t test and ANOVA. P < 0.05 was considered statis-
ACKNOWLEDGMENTS. We thank Robert Oshima and Helene Baribault for
providing the K8−/−mice; Evelyn Resurreccion for tissue sectioning and fluo-
rescence staining; Jean Chen, Alexander Hanganu, and Kris Morrow for
assisting with qPCR, colon homogenization, and figure preparation, respec-
tively; and Elisabeth Bik and Daniel DiGiulio for guidance on microbial DNA
qPCR. This work was supported by National Institutes of Health Grants R01
DK47918 (to M.B.O.), K08 DK069385 (to A.H.), DK56339 (to Stanford Univer-
sity), and DK34933 (to the University of Michigan), a European Union FP7
International Reintegration Grant, and grants from the Academy of Finland
and the Juselius Foundation (to D.M.T.).
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| www.pnas.org/cgi/doi/10.1073/pnas.1010833108Habtezion et al.