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Emerging Science
Claudins, dietary milk proteins, and intestinal
barrier regulation
Belinda M Kotler, Jane E Kerstetter, and Karl L Insogna
The family of claudin proteins plays an important role in regulating the intestinal
barrier by modulating the permeability of tight junctions. The impact of dietary
protein on claudin biology has not been studied extensively. Whey proteins have
been reported to improve intestinal barrier function, but their mechanism of action
is not clear. Recent studies, however, have demonstrated increased intestinal claudin
expression in response to milk protein components. Reviewed here are new
findings suggesting that whey-protein-derived transforming growth factor b
transcriptionally upregulates claudin-4 expression via a Smad-4-dependent
pathway. These and other data, including limited clinical studies, are summarized
below and, in the aggregate, suggest a therapeutic role for whey protein in diseases
of intestinal barrier dysfunction, perhaps, in part, by regulating claudin expression.
© 2013 International Life Sciences Institute
INTRODUCTION
Epithelial monolayers form selective barriers to protect
organ systems from the external environment. A critical
example is the gut epithelium, which has evolved complex
mechanisms not only to allow nutrient absorption but
also to defend against entry of infectious agents and
foreign antigens into the body. The impact of nutrients
on gut epithelial function and, in particular, the gut epithelial barrier, is poorly understood. Molecular mechanisms of nutrient actions in this tissue are also unclear,
but recent work has provided new insights into this area.
Absorption of nutrients, including minerals, can
occur through both transcellular and paracellular mechanisms. Transcellular mechanisms usually involve specific
cell-surface transporters or acceptor molecules that allow
for selective uptake of molecules that are transported
across the cell; this transport is often achieved with the
assistance of carrier proteins that are either utilized by the
intestinal epithelia or are exported through the basolateral membrane to the systemic circulation. In contrast,
paracellular transport involves the movement of molecules, usually across a concentration gradient, between
cells. The selectivity of paracellular transport relies on the
specific composition of the proteins that form intercellular junctions. This review focuses on paracellular transport and the role of tight junctions in that process.Within
an epithelial monolayer, cell-to-cell contacts, known as
tight junctions, regulate the flux of water, ions, and proteins across tissue barriers by allowing for the movement
of molecules between cells.1 Tight junctions are composed of membrane-spanning proteins, whose components traverse the paracellular space, and scaffolding
proteins, which link membrane proteins directly to the
actin cytoskeletal network. As shown in Figure 1,2
membrane-spanning proteins, including claudin, occludin, junction adhesion molecule, and scaffolding proteins, including the zona occludens, collectively regulate
the permeability of tight junctions.3 Claudins are essential
for barrier function by virtue of their critical role in
regulating selectively permeable ion channels in tight
junctions.4
Recently, the influence of nutrition on the biology
of tight junctions and, specifically, on the regulation of
activity and level of expression has become an area of
investigative interest. Although the effects of nutrients
Affiliations: BM Kotler and JE Kerstetter are with the Department of Allied Health Sciences, University of Connecticut, Storrs, Connecticut,
USA. KL Insogna is with the Department of Internal Medicine, Section of Endocrinology, Yale University, New Haven, Connecticut, USA.
Correspondence: BM Kotler, Department of Allied Health Sciences, University of Connecticut, 358 Mansfield Rd, Unit 2101, Storrs, CT
06269-2101, USA. E-mail: [email protected], Phone: +1-860-486-1996.
Key words: claudin, intestinal barrier function, TGFb, tight junction, whey protein
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doi:10.1111/j.1753-4887.2012.00549.x
Nutrition Reviews® Vol. 71(1):60–65
Figure 2 Claudin structure. A. Key features of a typical
claudin in the plane of the membrane. Shown are two
extracellular loop domains, where extracellular loop 1 (EL1)
contains a putative disulfide bond (S-S). Cylinders represent
predicted transmembrane alpha-helical domains. Also
illustrated are two palmitoylation motifs (P) and the
PDZ-binding motif at the extreme C-terminus. B. The four
transmembrane alpha-helical domains are depicted as a
tightly packed complex. Adapted from Overgaard et al.4
Abbreviation: COOH, C terminus.
Figure 1 Structure of tight junctions. Membranespanning proteins such as claudin, occludin, and junction
adhesion molecule (JAM) seal the paracellular space
between adjacent epithelial cells. Scaffolding proteins, such
as the zona occludens, act as adaptors that connect
membrane-spanning proteins to the actin cytoskeletal
network. Adapted from Ulluwishewa et al. 2
Abbreviations: ZO1/2, zona occludens 1 and 2; ZO1/2/3, zona
occludens 1, 2, and 3.
and of dietary protein in particular on claudin expression
and intestinal barrier regulation have not been studied
extensively, they are likely to be important, given the critical role of claudins in regulating the gut epithelial barrier.
This review focuses on recent studies that report an effect
of whey protein on claudin-4 expression and the impact
this has on intestinal barrier function.5
DESCRIPTION AND STRUCTURE OF THE
CLAUDIN FAMILY
The family of claudin proteins currently consists of 24
members. Claudins were first identified by Furuse et al.6
and derive their name from the Latin word “claudere,”
which means “to close.”3 Claudins have a molecular mass
ranging from 20 kDa to 27 kDa and are composed of four
transmembrane domains, two extracellular loops, a short
cytosolic N-terminus, and a longer cytosolic C-terminus
that includes a PDZ-binding domain, which is present in
nearly all isoforms (Figure 2).4 Extracellular loop 1 (EL1)
consists of approximately 52 amino acids, including a
conserved cysteine-containing motif,4,7 and is predominantly responsible for the charge-selective permeability
of paracellular transport. Extracellular loop 2 (EL2),
approximately 16 to 33 amino acids long, is presumed to
Nutrition Reviews® Vol. 71(1):60–65
fold into a helix motif and, along with EL1, is critical
for claudin-claudin interaction.4 The length of the
C-terminus varies between claudin isoforms, from 21 to
63 amino acids, and contributes to isoform-dependent
paracellular selectivity.3 The C-terminus PDZ-binding
motif facilitates direct interaction between claudins and
scaffolding proteins. Not all claudins contain a PDZbinding domain, claudin-12 being an example.8 The
N-terminus consists of seven amino acids and has no
known function.1
CLAUDIN FUNCTION
As noted above, intestinal absorption of nutrients can
occur via transcellular or paracellular transport. In contrast to transcellular transport, which usually requires
energy expenditure, paracellular transport is passive and
depends on an electrochemical gradient. The tight junctions, including the claudin proteins, facilitate paracellular transport by forming ion-selective pores.9
Claudins can be classified as either pore forming or
pore sealing. Pore-forming claudins increase paracellular
permeability through the formation of channels, whereas
pore-sealing claudins reduce paracellular permeability. A
wide variety of incompletely understood factors determine whether claudins enhance or reduce paracellular
permeability. These include claudin-claudin interactions,
which can occur head-to-head between the same claudins
on adjacent cells (homotypic interactions) or, much less
frequently, between different claudins (heterotypic interactions). Claudins can also interact side-to-side in homomeric or heteromeric interactions4 (see also the citations
in Overgaard et al.4). Additionally, claudins may interact
with other components of the tight junction, including
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occludins and zona occludens 1, and can undergo posttranslational modification such as palmitoylation and
serine-threonine phosphorylation.4 These complex interactions in the microenvironment of the tight junction, as
well as the tissue-specific expression of certain claudins,
underlie their varied effects on permeability, which are for
the most part poorly understood.
Based in part on their molecular size, solutes are
thought to cross tight junctions. Solutes less than 4 angstroms (e.g., sodium, magnesium) follow a pathway that
carries electrical charge during transport. Increased
claudin-2 expression in cultured epithelia increases permeability for solutes less than 4 angstroms. The pathway
for solutes greater than 4 angstroms (e.g., mannitol,
inulin) carries no electrical charge and is speculated to
represent temporary breaks in otherwise continuous tight
junctions.10
Charge-selective regulation of claudin proteins is
determined by the structural elements in EL1. The negatively charged residues in EL1 strongly influence cation
pore formation. For example, the replacement of basic
residues with acidic residues in the EL1 domain of
claudin-4 increased cation permeability. EL2 collaboration with EL1 seems to be required for proper pore
formation.11
CLAUDINS AND INTESTINAL BARRIER REGULATION
Several claudins have been reported to have functional
roles in the intestine. Claudin-1, -3, -4, -5, and -8 function
as pore-sealing proteins, whereas claudin-2 forms chargeselective pores, for example, facilitating the paracellular
transport of calcium. Claudin-12 has also been reported
to increase paracellular calcium transport in enterocytes.12 Claudin-7 appears to reduce tight-junction permeability, as indicated by studies in knockout mice.13
The role of claudin-15 in regulating intestinal barrier
function is unclear, but this claudin seems to have distinct
biological actions in enterocytes, where it increases cell
proliferation.14
Paracellular permeability can inappropriately
increase with disease and age, as well as during periods of
stress. Understanding the mechanisms by which claudin
proteins regulate the intestinal barrier will provide
insight into the pathophysiology of intestinal barrier malfunction as well as identify targets for therapeutic intervention.2 Consistent with this idea, recent studies indicate
that manipulation of claudin function in experimental
models affects the intestinal barrier. Ewaschuk et al.15
determined that claudin-2 expression is suppressed in
T84 cell monolayers (an enterocyte cell model) following
4 hours of treatment with metabolites secreted by the
commensal organism Bifidobacterium infantis. Thus, one
mechanism by which microorganisms could affect intes62
tinal permeability is by altering the expression of claudins. Suzuki and Hara16 discovered that the most
common flavonoid present in nature, quercetin, decreases
paracellular flux across Caco-2 cells and increases
claudin-4 protein expression. Yeh et al.17 determined that
chitosan, a widely used food additive, increases paracellular flux in Caco-2 cells. During the postexposure period,
when tight-junction function is reestablished, the investigators found that claudin-4 transcription increased.
Taken together, the above findings15–17 suggest that
claudin activity and expression can be dynamically
altered by the diet and by changes in the intestinal
microbiota.
MILK PROTEIN COMPONENTS AND INTESTINAL
CLAUDIN EXPRESSION
Recent work by Hering et al.5 adds to the heretofore
limited understanding of how nutrition influences
claudin biology. The investigators focused on whey, the
so-called “fast-digested milk protein,” specifically whey
protein concentrate 1 (WPC1). Consistent with earlier
studies in both humans and rodents,18,19 these investigators found that WPC1 protected against an interferon
gamma (IFNg)-induced tight-junction barrier disturbance. They explored the molecular mechanisms by
which whey protein, and WPC1 in particular, exert this
effect. Milk, whey protein, and WPC1 all contain high
levels of transforming growth factor b1 (TGFb1), which
has been reported to improve intestinal tight-junction
function.18 To determine if TGFb1 mediates the effects of
WPC1 on intestinal tight junctions and to clarify the
molecular mechanism by which TGFb1 acts, the authors
first showed that WPC1 and TGFb1 both increased transepithelial resistance. To explore the downstream signaling
cascade that mediates this effect, the authors next investigated known TGFb1 signaling intermediaries. It had
been previously reported that milk and TGFb activated a
canonical TGFb reporter construct.18 However, it had
also been suggested that Smad-independent pathways
mediated the effects of TGFb on epithelial tight junctions.20 As noted, claudin-4 has been previously reported
to regulate tight-junction function. Interestingly, these
investigators found that WPC1 and TGFb1 increased
expression of claudin-4 but did not affect the expression
of claudin-1, -2, -5, -7, or occludin. They therefore focused
on claudin-4 and used a claudin-4 promoter construct to
evaluate cell-signaling pathways entrained by WPC1 and
TGFb1. As compared with nonfat dried milk, which has
less TGFb1, WPC1 activated the claudin-4 promoter
more effectively. TGFb1 was also able to induce this construct. This activation was significantly enhanced by overexpression of Smad-4, a transcription factor known to be
a target of TGFb1.21 Deletional analysis, as well as mutaNutrition Reviews® Vol. 71(1):60–65
tion of the Smad-4 binding site of the claudin-4 promoter, established that Smad-4 binding was required for
the ability of both TGFb1 and WPC1 to activate the promoter. Thus, HT-29/B6 cells treated with TGFb1
(60 ng/L) for 48 hours showed an increase in the activity
of the claudin-4 promoter. Overexpression of Smad-4
augmented this effect, while mutation of the Smad-4
binding site in the claudin-4 promoter abolished the
ability of TGFb1 to induce claudin-4 promoter activity.
To determine if TGFb1 mediated the effects of WPC1 on
claudin-4 transcription, cells were transfected with a
claudin-4 promoter and treated with WPC1 in the presence of neutralizing antibodies to TGFb1, 2, and 3. These
neutralizing antibodies blocked the stimulatory effect of
WPC1 on the promoter.
Hering et al.5 concluded that WPC1 mediates its
action on intestinal barrier function in part by inducing
claudin-4 expression via a TGFb/Smad-4 signaling
cascade. These studies provide a plausible molecular
mechanism by which milk and whey protein exert their
salutary effect on intestinal epithelial integrity. While
these models of overexpression support a role for TGFb
in Smad-4-mediated regulation of tight junctions,
complementary studies involving knockdown of endogenous Smad-4 would provide key additional support for
this model.
The potential clinical relevance of this proposed
molecular pathway is supported by in vivo evidence that
TGFb has beneficial effects on the intestinal barrier. In a
mouse model, oral administration of 500 mL of cow milk
containing TGFb (3,000 ng/L) daily for 2 weeks before
induction of colitis and endotoxemia ameliorated both
tissue damage and mortality.18 In the neonate, TGFb has
been found to be important for gut homeostasis and
regulation of inflamation.22 Hering et al.5 suggest that
TGFb1 in concentrations as little as 60 ng/L can be beneficial to mucosal restitution. The WPC1 used by Hering
et al.5 contained 30–60 ng/g of TGFb1.Although the dilutional effect of gastric and intestinal secretions on
ingested WPC1 is not known, it seems reasonable to
assume that the ingestion of several grams of WPC1
would result in luminal TGFb1 concentrations in the
60 ng/L range. It is plausible that ingestion of WPC1
could induce biologically significant effects on intestinal
barrier protection. Thus, TGFb1-rich WPC1 may have
clinical efficacy in gastrointestinal disorders characterized by barrier dysfunction. This is especially appealing in
the neonate, in whom the dilution factor between the
mouth and duodenum is less than in the adult.
In addition to whey protein, casein, the most abundant protein in milk, may influence barrier function and
claudins. Visser et al.23 reported that a high-casein diet
improved intestinal barrier function in diabetes-prone
BioBreeding (DP-BB) rats. At weaning, DP-BB rats were
Nutrition Reviews® Vol. 71(1):60–65
placed on a high-casein (HC) diet (containing 200 g/kg
casein) or a standard plant-based diet for 65 days. Rats on
the HC diet had improved intestinal barrier function as
assessed by measuring the ratio of lactulose to mannitol
in the urine after oral administration of lactulose and
mannitol.23 Serum zonulin levels, which correlate
strongly with intestinal permeability, were lower in the
animals on the HC diet.23 The HC animals also had
increased transepithelial resistance as compared with
animals on the standard diet. Quantitative polymerase
chain reaction was performed using RNA isolated from
the ileum of the HC animals to investigate the effect of the
diet on expression of tight-junction proteins. Rats on the
HC diet had increased expression of pore-sealing
claudin-1 and a reduced expression of pore-forming
claudin-2.23 Interestingly, Hering et al.5 found that caseinbased diets contain little TGFb1. These data suggest that
the HC diet reduces intestinal permeability in this experimental rat model by a TGFb1-independent mechanism.
While the anti-inflammatory effects of TGFb within
the intestine have long been known24,25 and are mediated,
in part, by modulation of the relative proportions of proand anti-inflammatory CD4+ T cell subsets, these data
support an alternative mechanism. The studies of Hering
et al.5 and Visser et al.23 are consistent with the idea that
milk protein components improve intestinal barrier
function, in part, by altering the expression and functions
of claudins via both TGFb1-dependent and TGFb1independent pathways. In support of this hypothesis, previous reports demonstrate that a variety of intestinal
diseases characterized by altered permeability are accompanied by changes in claudin expression.26,27
ALTERED CLAUDIN EXPRESSION IN
INTESTINAL DISEASE
As many as 1.4 million Americans suffer from inflammatory bowel disease.28 Altered intestinal barrier function is
a common feature of the major inflammatory bowel diseases, Crohn’s disease and ulcerative colitis, as well as
diseases such as collagenous colitis. Increased antigen
uptake into the circulation can initiate and intensify epithelial inflammation, while water uptake from the circulation to the intestinal lumen can result in diarrhea. This
can lead to an auto-intensifying cycle of disease in which
increased permeability results in increased antigen
uptake, which further worsens the inflammation and
diarrhea.
Zeissig et al.27 recently reported changes in claudin
expression in colonic biopsies from patients with Crohn’s
disease. Expression of pore-sealing claudin-5 and
claudin-8 was downregulated, while expression of poreforming claudin-2 was strongly upregulated. Claudin-5
and claudin-8 were also found to redistribute away from
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the tight junction, as determined by immunohistochemistry. In addition to the changes in the molecular composition of the tight junction, the rate of epithelial cell
apoptosis was also found to be upregulated.
Alterations in tight-junction structure and function
have also been observed in ulcerative colitis. Expression
of pore-forming claudin-2 is upregulated, accompanied
by an increase in the rate of epithelial cell apoptosis,
resulting in epithelial lesions. In collagenous colitis, intestinal barrier regulation is significantly altered, with downregulation of pore-sealing claudin-4 and upregulation of
pore-forming claudin-2 observed in some patients.26
Interestingly, and in contrast to the findings in Crohn’s
disease and ulcerative colitis, rates of epithelial cell apoptosis were not increased in collagenous colitis.
Since downregulation of claudin expression has been
reported to occur frequently in inflammatory bowel
disease and since TGFb upregulates expression of at least
claudin-4, it could be postulated that TGFb might serve as
a potential therapeutic agent in inflammatory bowel
disease, in part by helping to reestablish intestinal barrier
function via its effect on claudins. Consistent with that
notion, Fell et al.29,30 have reported that a polymeric diet
enriched in TGFb induced healing in pediatric patients
with Crohn’s disease.
CONCLUSION
Tight junctions, including the claudin proteins, are a
crucial component of the intestinal barrier responsible
for integrity and permeability. The varied effects of claudins on permeability are, for the most part, poorly understood. It has been suggested that claudin function, and
thereby tight-junction permeability, can be dynamically
altered by the diet and changes in the intestinal
microbiota.15–17 Specifically, Hering et al.5 and Visser
et al.23 suggest milk protein components improve intestinal barrier function, in part, by altering the expression
and functions of claudins. These studies5,23 are limited by
the fact that animal models exclusively were studied,
leaving uncertain the relevance of these findings in
humans. The reliance on overexpression studies to establish the role of Smad-4 in the TGFb1/claudin-4 pathway
by Hering et al.5 requires that additional studies be performed before this signaling cascade can be accepted as
broadly applicable in vitro and in vivo.
Nonetheless, the studies summarized above5,18–23 add
to the current understanding of how nutrition influences
barrier regulation. Several mechanisms contribute to
intestinal barrier dysfunction and disease. Investigation
of tight-junction protein regulation of intestinal disease
may provide new targets for therapeutic intervention.
Further research is necessary to confirm the mechanisms
by which milk protein components improve intestinal
64
integrity, as well as whether milk protein components
should be considered as a therapy for diseases characterized by intestinal barrier compromise. Clinical trials
using whey-protein isolates in humans with intestinal
barrier dysfunction would be of considerable interest. In
addition to noninvasive assessment of intestinal barrier
function, intestinal mucosal biopsies to assess claudin
expression would be of interest. If well-designed and
properly controlled, such studies would significantly aid
in determining whether claudins deserve attention as
potential therapeutic targets, and, if so, which ones should
be pursued.
Acknowledgment
All listed authors contributed significantly to the work’s
conception, design, data collection, or data interpretation
and analysis; participated in the writing or critical revision of the article in a manner sufficient to establish ownership of the intellectual content; and read and approved
the version of the manuscript being submitted.
Funding. This publication was made possible in part by
grant number 460712 from the University of Connecticut
(to BMK). Its content is solely the responsibility of the
authors and does not necessarily represent the official
view of the University of Connecticut. This work was also
supported by a grant from the United States Department
of Agriculture (USDA 2009-65200-04920, to KLI and
JEK).
Declaration of interest. The authors have no potential
conflicts of interest to declare.
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