For organisms composed of a sphere of cells, growth is limited
by the diffusion of oxygen. This was the likely reason for the
appearance of epithelia, which allowed organisms to trap a
small portion of the primitive oceans and to alter its
composition to make the first milieu interieure. Hence, along
with germ cells, epithelia must have been one of the first
differentiated cell types to evolve in multicellular organisms.
Genetic analysis suggests that sponges might represent the first
metazoans, although some researchers have proposed that
Trichoplax is closer to the ‘Urmetazoan’ than are sponges
(Schierwater, 2005). Sponges and Trichoplax have a simple
body plan, containing only a few types of epithelial and germ
cells. A brief look at the catalogue of cell types in humans
reveals a few hundred cell types, perhaps as many as three
quarters of them being epithelia.
During embryonic development, the rudiments of most
organs contain what look like generic epithelia, flat cells that
are connected by tight junctions and adherens junctions rest on
a basement membrane and exhibit apical-basal polarity. We can
term these cells proto-epithelia in part because all other
epithelia originate from them. In mature organs, the shape of
cells is quite different and characteristic of the organ in
question. There are ‘simple’ epithelia, i.e. epithelia composed
of a sheet of single cells. Some of these are squamous (such as
endothelia), others are cuboidal (e.g. in the kidney tubules) or
columnar (e.g. in the intestine). There are also multi-layered
epithelia such as transitional (e.g. urinary bladder) and
stratified squamous epithelia (e.g. skin).
In some cases these terminally differentiated cells and their
less differentiated precursors exist simultaneously. In the small
and large intestine, the epithelial sheet is organized into
characteristic folds to form crypts and villi. Crypts contain
stem cells that can generate all four types of epithelial cells of
the intestine (Marshman et al., 2002). In the prostate, basal
cells also thought to be stem cells co-exist with luminal cells
that are their differentiated progeny. Similarly in the skin, basal
cells generate the squamous epithelium that is destined to be
cornified. In each organ, the morphology of the less
differentiated cell is clearly different from that of the terminally
differentiated one and their gene expression patterns of course
differ. It is difficult to define characteristics common to all
terminally differentiated epithelia but not their precursors.
However, at least in simple epithelia, we can provide a
preliminary list. Perhaps the most obvious difference is in the
apical compartment of the cell. For instance, embryonic
pancreatic acinar cells or mucus secreting goblet cells have no
secretory granules but fully differentiated mature ones do.
Another obvious difference is the presence of brush border
microvilli in the intestine: crypt cells have few microvilli
whereas the absorptive epithelia of the villus (or surface in
the colon) have exuberant microvilli. Microvilli contain
cytokeratins, actin and actin-binding proteins such as villin.
Although some crypt cells express these proteins, their
organization into a subapical terminal web is a characteristic
of absorptive cells.
Since all of these cell types originate from proto-epithelia,
one can posit a pathway of epithelial differentiation that goes
from proto-epithelia to terminally differentiated epithelia.
Defining this pathway is an important goal, because many
cancer biologists believe that a block in terminal differentiation
of epithelial cells can be a root cause of cancer. Various
Epithelia, the most common variety of cells in complex
organisms exist in many shapes. They are sheets of
polarized cells that separate two compartments and
selectively transport materials from one to the other. After
acquiring these general characteristics, they differentiate to
become specialized types such as squamous columnar or
transitional epithelia. High density seeding converts a
kidney-derived cell line from flat ‘generic’ epithelial cells
to columnar cells. The cells acquire all the characteristics
of differentiated columnar cells, including microvilli, and
the capacity for apical endocytosis. The high seeding
density induces the deposition of a new protein termed
hensin and polymerization of hensin is the crucial event
that dictates changes in epithelial phenotype. Hensin is
widely expressed in most epithelia. Its deletion in mice leads
to embryonic lethality at the time of generation of the first
columnar epithelium, the visceral endoderm. Moreover
many human cancers have deletions in the hensin gene,
which indicates that it is a tumor suppressor.
Key words: Hensin, DMBT1, Epithelial terminal differentiation,
Differentiation of columnar epithelia: the hensin
Soundarapandian Vijayakumar1, Jiro Takito1,*, XiaoBo Gao1, George J. Schwartz2and Qais Al-Awqati1,‡
1Departments of Medicine and Physiology and Cellular Biophysics, College of Physicians and Surgeons of Columbia University, 630 W 168th St,
New York, NY 10032, USA
2Department of Pediatrics, University of Rochester School of Medicine, Rochester, NY, USA
*Present address: Keio University, Tokyo, Japan
‡Author for correspondence (e-mail: email@example.com)
Accepted 20 September 2006
Journal of Cell Science 119, 4797-4801 Published by The Company of Biologists 2006
Journal of Cell Science
molecules play a role, including members of the Wnt, FGF
and BMP gene families. Here we discuss a key driver of
this process, the extracellular matrix protein hensin.
Intercalated cells in the kidney
The collecting tubule of the nephron is composed of two
epithelial cell types: the majority are principal cells
responsible for transport of Na+and water; and the
remainder are the intercalated cells dedicated to H+
transport. Recent studies have shed significant light on the
mechanism determining the epithelial cell phenotype. At
one extreme is the ?-intercalated cell, which has an apical
Cl–:HCO3–exchanger and a basolateral H+ATPase; hence
this cell secretes HCO3–into the lumen of the tubule. The
other extreme is the ?-intercalated cell in which the H+
ATPase is located in the apical membrane whereas the Cl–:
HCO3–exchanger is located in the basolateral membrane.
Remarkably, the ?-intercalated cell converts to an ?
phenotype when the tubule segment (or the animal in vivo)
is exposed to an acid medium (Schwartz et al., 1985). The
trans-epithelial transport of this segment also reverses
direction from secretion of HCO3–to secretion of acid, thereby
playing an important role in the regulation of the pH of the
The ?-intercalated cell can be established as an
immortalized clonal cell line. Confluent epithelia formed by
cells from this line secrete HCO3–from the apical side. Seeding
them at low density on filters generates flat, large cells with
minimal surface microvilli. These show no apical endocytosis
and have no actin network underneath the apical membrane.
Remarkably, when the same cells are seeded at high density
and allowed to proceed to confluence, they assume a
completely different phenotype: they form columnar cells that
are twice as tall and develop robust apical endocytosis and a
thick sub-apical actin cytoskeleton. The low-density cells are
‘real’ epithelia; i.e. they have polarized apical and basolateral
membranes that contain different proteins (van Adelsberg et
al., 1994) and lipids (van’t Hof et al., 1997). They contain
lateral and tight junctions but have no sub-apical actin and do
not express villin or cytokeratin 19. By contrast, the high-
density cells have sub-apical actin, and express cytokeratin 19
and villin (Vijayakumar et al., 1999) (Fig. 1). In vivo, ?-
intercalated cells have vigorous apical endocytosis whereas the
? form cells have none. The ? form has essentially no
microvilli whereas the ? form exhibits prominent apical folds
and microvilli. In addition, examination of electron
micrographs of the cortical collecting tubule shows that ?-
intercalated cells are taller, they jut into the tubule lumen
whereas the ? forms are more flat. High seeding density thus
induces a transformation of ?-intercalated cells similar to that
seen in vivo when they convert into ?-intercalated cells. Note
that all of these studies were performed in a cell culture system
where the inducing event is seeding cells at high density.
During epithelial differentiation in vivo, all cells touch each
other and hence they are at a constant density. Hence, the high
density seeding must be spuriously activating a pathway that
is normally acting using another signaling event.
Hensin, a phenotypic regulator of epithelial cells
How does simple seeding at high density cause this
transformation? A factor located in the extracellular matrix of
Journal of Cell Science 119 (23)
the high density cells converts them to columnar cells. This can
be captured on filters and makes low-density cells acquire the
same features as high density cells; they become taller and have
sub-apical actin and also begin to express cytokeratin 19 and
villin Fig. 1. The protein responsible for this change has been
purified and is termed hensin.
Hensin is a 180 kDa secreted glycoprotein that is expressed
in all epithelia tested (Takito et al., 1999). It has a large number
of alternatively spliced isoforms that vary in size up to an
apparent molecular weight of 340 kDa (Fig. 2). In many
organs, the more caudal a tissue, the higher is the level of
expression; for instance, in the gastro-intestinal tract the
highest level of expression is in the colon. In the kidney, hensin
is expressed only in the ureteric bud lineage (also caudal) but
there is no expression in the epithelia derived from the
metanephric mesenchyme. Hensin is also expressed in the
brain, in the lung, in the skin and even in macrophage
derivatives such as osteoclasts.
Hensin contains three types of domain: SRCR (scavenger
receptor cysteine rich), CUB (Clr-Uef-BMP1) and Zp (zona
Fig. 1. Distribution of cytoskeletal proteins in low-density and high-
density cells. X-Z Optical sections of the intercalated cell line cultured
at low density, high density and at low density but on the extracellular
matrix of high-density cells. The cells were stained by phalloidin and
with antibodies to villin and cytokeratin 19. Note the cell height in
high-density cells and in cells seeded on high-density matrix.
Fig. 2. Domain structure of hensin isoforms.
Journal of Cell Science
Hensin and epithelial differentiation
pellucida) (Fig. 2). The most abundant are the SRCR domains,
which comprise 100-110 residues and are present in a large
number of proteins in all metazoans, including sponges; but no
specific function has been assigned to them (Sarrias et al.,
2004). CUB domains are also ~110 residues long and were first
identified in the complement components C1r and C1s. They
are present in a large number of proteins but remain of
unknown function (Bork and Beckmann, 1993). Zp domains
(for Zona pellucida) were first identified as sperm receptors and
are composed of about 280 residues. They are present in a wide
array of organisms and proteins and they are thought to be
involved in protein polymerization (Jovine et al., 2005).
The genomic sequence of hensin encodes 18 SRCR
domains, 6 CUB domains and 1 ZP region, as well as a
transmembrane domain. Each epithelium seems to express a
specific splice form. The cDNAs of some of these isoforms
reveal the presence of a membrane anchor, but in the intestine
where this sequence is present all hensin forms are nevertheless
secreted (Cheng et al., 1996); in other cases (such as the
salivary form gp340) (Holmskov et al., 1999), hensin lacks the
anchor and clearly is secreted. There have been some
suggestions that secreted forms of hensin play a role in immune
defense by binding to bacteria (Madsen et al., 2003),
surfactants or trefoil factors (Thim and Mortz, 2000). However,
most of these are based on in vitro binding studies, and given
that many of the domains of hensin are protein-protein
interaction domains it is difficult at present to decide whether
these studies actually reveal physiological roles of hensin.
Antibodies that recognize SRCR domains block the ability of
hensin to induce formation of columnar epithelia, which
suggests that these domains play a crucial role in either binding
to receptors or to the formation of the hensin fibril (see below).
Polymerization of hensin
Remarkably, the low-density cells abundantly synthesize
hensin and secrete it in a polarized manner from the basolateral
side. However, they secrete only monomers of hensin whereas
high-density cells produce an array of hensin forms from
monomers to very-high-order polymers, only the highest order
of which precipitate in the ECM (Hikita et al., 1999). Studies
suggested that only these insoluble forms of hensin in the ECM
are capable of converting cells displaying the low-density
phenotype to columnar epithelia.
Thus, like other extracellular matrix proteins, hensin is
synthesized as a soluble monomer but it is only the multimers
that deposit in the basement membrane and mediate its
functions. Some ECM proteins such as collagen need to be
specifically proteolyzed to generate the insoluble forms. Others,
such as fibronectin, require the cell to participate in the formation
of the complex. In the case of hensin, at least three other proteins
are required to form the active multimers. The first one identified
was galectin 3. Galectin is a member of a large family of lectins
that bind to ?-galactosides. It can convert hensin monomers to
dimers. Moreover, extraction of galectin 3 from insoluble hensin
removes its ability to convert cells to columnar epithelia and
adding galectin3 back restores this ability (Hikita et al., 2000).
This suggests that galectin 3 not only stimulates formation of the
hensin fibril, perhaps by making hensin dimers, but also might
maintain these multimers in the correct orientation. Galectin 3
unlike other galectins contains a protein-protein interaction
domain in addition to the carbohydrate-recognition domain.
Hence, it is likely to induce multimeric association of
glycoproteins. Interestingly the galectin-3-knockout mice are
viable and fertile and do not seem to have any anomalies in
epithelial organs (Colnot et al., 1998). All galectins are defined
by the presence of homologous carbohydrate-binding domains;
hence if their role in hensin polymerization is mediated by
binding of the lectin to carbohydrate moieties, other members of
this large family of proteins could substitute for galectin 3 in
A cis-trans peptidyl prolyl isomerase is also necessary for
hensin polymerization and this could be a member of the
cyclophilin family of prolyl isomerases (Watanabe et al., 2005).
In addition, recent studies suggest that integrins need to be
activated and that tyrosine phosphorylation of ?1 integrin is
crucial (our unpublished results). The order in which these three
proteins act in hensin polymerization is not yet clear.
Conversion of ? ?- to ? ?-intercalated cells is mediated
The collecting tubule is a major site of regulation of acid-base
transport in the kidney. When animals are fed an alkaline ash
diet, it secretes HCO3–into the lumen. Feeding these animals
an acid diet reverses the direction of the flux to absorption. This
is accompanied by a decrease in the number of ?-intercalated
cells that secrete HCO3–and an increase in the number of acid-
secreting ?-intercalated cells, which absorb HCO3–. By
measuring the intracellular pH of individual ?-intercalated
cells, one can demonstrate that the same cells that secrete
HCO3–through an apical Cl–:HCO3–exchanger before acid
exposure begin to secrete acid and develop a basolateral
Cl–:HCO3–exchanger. Since the cells internalize the apical
exchanger, they must also develop apical endocytosis
(Schwartz et al., 2002). The cells that change their phenotypic
state in this way also start laying down hensin in the
extracellular matrix. Addition of anti-hensin antibodies
prevents the acid-induced changes in the location of the
Cl–:HCO3–exchangers. Hensin antibodies also prevent the
switch from the HCO3–secretion to HCO3–absorption seen on
exposure to acid. Hensin thus not only can cause formation of
columnar epithelia in culture but also drive a physiologically
relevant change in the phenotype of intercalated cells. It is
interesting that examination of published electron micrographs
of ? and ? intercalated cells shows that the ? intercalated is
much taller than the ? form suggesting that this process is also
a columnarization event.
General role of hensin in differentiation in vivo
Since hensin is broadly expressed in various epithelial cells, it
might play a general role controlling their phenotype. In the
intestine, for example, proliferating cells that ascend the crypt
wall differentiate as they go towards the surface. As they reach
the villi (in the small intestine) or the surface (in the colon)
they become terminally differentiated, acquiring all the
characteristics of absorptive intestinal cells, including
microvilli and a variety of markers of specific markers of
differentiation (Crosnier et al., 2006). Hensin in differentiated
cells is present in the villus, adopting a pattern similar to that
of extracellular matrix proteins. By contrast, in the crypt cells
it is cytoplasmic. Generation of conditional knockout alleles
should reveal the details its role in these tissues, as well as the
functions of hensin in other epithelia.
Journal of Cell Science
Hensin in cancer
Screens of a variety of human tumors have revealed that hensin
is deleted in the majority of epithelial cancers. Much of this
work was based on the discovery that certain malignant brain
tumors lack the gene encoding DMBT1 (deleted in malignant
brain tumor 1) (Mollenhauer et al., 1997), which was later
found to be a human orthologue of hensin (Takito et al., 1999).
The proportion of tumors lacking hensin/DMBT1 varies, in
glioblastomas as many as half of them exhibit it, whereas in
others the incidence is lower. Deletions have been found in
skin, breast, oral, esophageal, colon and lung cancers by a
variety of methods. Some of these studies have used cancer cell
lines, but most also included resected tumors. There is a
general idea in cancer biology that tumorigenesis requires that
terminal differentiation of the epithelia be abrogated and,
hence, the widespread deletion of hensin/DMBT1 in several
tumors suggests it might be a tumor suppressor gene.
Hensin in embryonic development
A global deletion of hensin is embryonic lethal. Homozygous
mutants fail to survive beyond embryonic day (E) 4.5, but
blastocysts (E3.5) are obtained in appropriate mendelian
proportions. Incorporating ?-galactosidase into the hensin
locus reveals that hensin begins to be expressed in the
trophectoderm and is present at a low level in the embryonic
stem (ES) cells. A few hours later the primitive endoderm
begins to express hensin at a high level. This is a layer of
epithelial cells that covers the ES cells in late blastocyst
development; it eventually forms the visceral endoderm, an
epithelial layer that surrounds the whole embryo, and parts of
it form the placenta (Fig. 3). The first cells that express hensin
are these extra-embryonic tissues. The primitive endoderm is
crucial for assuring the normal development of ES cells into
the embryo proper. In hensin-deficient mice it has a large
number of apoptotic cells. The death of the embryo might thus
be due to a defect in ES cell survival resulting from abnormal
primitive endoderm development. Culturing blastocysts in
vitro confirms this: the mutant embryos all degenerate whereas
those taken from the wild-type and heterozygous mice survive
(Takito and Al-Awqati, 2004).
Once the blastocyst is implanted, the ES cells begin to divide
and the embryo elongates to form the egg cylinder stage.
Hensin is expressed in the visceral endoderm (VE). The top
part of this is the extra-embryonic VE. The bottom half is the
embryonic VE, which regulates the epiblast – i.e. the embryo
proper (Fig. 4). The extra-embryonic VE is a columnar
epithelium. The embryonic VE is a squamous epithelium
except at the tip, where a few cells are columnar. Hensin is
expressed only in the columnar epithelial cells. Remarkably,
these few columnar cells (distal VE) migrate upwards and
within a few hours become squamous epithelia (anterior VE)
and cause that region of the epiblast to become the head
(Srinivas et al., 2004). Blocking the movement of VE by
knocking out the transcription factor Hex, which is normally
expressed in these cells, results in truncation of the head
elements (Martinez-Barbara, 2000).
Hensin might thus be responsible for generation of columnar
epithelia in the early embryo in vivo. ES cells seeded on filters
coated with fibronectin and collagen IV grow as a monolayer
expressing some epithelial proteins but not in a pattern that
suggests polarized differentiation. By contrast, when grown on
laminin and hensin, the ES cells adopt a morphogenetic pattern
similar to embryoid bodies, forming hemi-spheres on the
filters. The surface epithelium of the hemi-spheres is covered
by a polarized epithelium that exhibits an apical cytoskeleton
composed of actin, cytokeratin 19 and villin. The surface
epithelia also display vigorous apical endocytosis, whereas ES
cells grown on fibronectin and collagen IV do not internalize
the marker (Takito and Al-Awqati, 2004).
These studies suggest that seeding ES cells on hensin or
laminin results in a differentiation event. However, the surface
epithelium of the hensin-grown hemispheres is columnar and
thus similar to the intercalated cell line described above.
Remarkably, the cells on the surface of the hemispheres seeded
on laminin are instead flat, low and squamous. Both of these
epithelia (but not those seeded on collagen IV or fibronectin)
express VE markers such as BMP2 and transthyretin. However,
cells seeded on laminin express ?-fetoprotein, another marker
of the VE. Another difference between these two types of
epithelia is the expression of desmosomal proteins.
Desmocollin is expressed in hemispheres grown on hensin;
those grown on laminin instead express desmoglein. In
embryos at the egg cylinder stage desmoglein is seemingly
expressed in all VE cells. Note also that the hemispheres
seeded on laminin are easily detached whereas those seeded on
hensin are much more firmly attached. Since the columnar
epithelia in the distal VE migrate to form the anterior VE,
which is rather squamous during migration, the cells might
switch signaling from hensin polymers to laminin polymers?
If so, how does this occur? They could degrade hensin and lay
down laminin or replace one receptor by another or alter some
other signaling event.
The study of the biology of hensin promises to uncover a
crucial aspect of epithelial cell biology. The conversion of
proto-epithelium to a differentiated epithelium under the
Journal of Cell Science 119 (23)
Fig. 3. Development of the mouse embryo between E3 and E4. The
upper panels are a schematic of the blastocyst development. A layer
of cells (in red) covering the ES cells (orange) becomes the
epithelium of the primitive endoderm. Later, some of its cells will
populate the inside layer of the trophectoderm to form the parietal
endoderm whereas that covering the ES cells will form the visceral
endoderm. In the lower panels are embryos taken from mice with
LacZ inserted into the hensin locus. The embryo on the right was
removed at E3.5 and cultured for 24 hours in vitro. Hensin
expression begins in the ES cells (E3.5) and eventually is
concentrated in the primitive endoderm.
Journal of Cell Science
4801 Download full-text
Hensin and epithelial differentiation
influence of hensin is probably the first such pathway
discovered. Because hensin is also expressed in the skin
(squamous epithelia), the urinary bladder and ureter
(transitional epithelium), it may be involved in a more
fundamental process of differentiation than conversion of
simple proto-epithelia into columnar epithelial cells. That
hensin seems to be deleted in a variety of epithelial cancers
suggests that it has a dominant role in forcing the epithelium
to maintain its differentiated state. All of these studies suggest
that hensin plays a general role in epithelial differentiation;
hence the identification of the signaling pathways involved
acquires a new urgency. Hensin signaling can be divided into
that which causes polymerization of hensin and that which
involves binding of polymeric hensin to its receptor. A central
question is whether hensin is central to a common pathway of
differentiation or is a common intermediate in a more diverse
set of pathways.
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Fig. 4. Epithelial types in the egg
cylinder stage. (A) A model of the
cell types in the egg cylinder. In the
visceral endoderm (VE) only the
distal VE and the extra-embryonic
VE are columnar epithelia, the rest
are flat and squamous looking. The
distal VE migrates to establish the
anterior VE. As found by Srinivas et
al. (Srinivas et al., 2004), its shape
changes from columnar to squamous
during the migration. (B) Expression
of hensin (in blue) in the egg cylinder
stage. Note that the embryo lacks
Journal of Cell Science