ADAM-15 inhibits wound healing in human intestinal

epithelial cell monolayers

Laetitia Charrier, Yutao Yan, Adel Driss, Christian L. Laboisse, Shanthi V.
Sitaraman and Didier Merlin
Am J Physiol Gastrointest Liver Physiol 288:G346-G353, 2005. First published 9 September 2004;
doi:10.1152/ajpgi.00262.2004

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Am J Physiol Gastrointest Liver Physiol 288: G346 –G353, 2005.
First published September 9, 2004; doi:10.1152/ajpgi.00262.2004.
ADAM-15 inhibits wound healing in human intestinal
epithelial cell monolayers
Laetitia Charrier,1 Yutao Yan,1 Adel Driss,1 Christian L. Laboisse,2
Shanthi V. Sitaraman,1 and Didier Merlin1
1
Department of Medicine, Division of Digestive Diseases, Emory University School of Medicine, Atlanta, Georgia;
and 2Faculte de Medecine, Institut National de la Santé et de la Recherche Médicale U539, Nantes, France
Submitted 17 June 2004; accepted in final form 2 September 2004
Charrier, Laetitia, Yutao Yan, Adel Driss, Christian L.
Laboisse, Shanthi V. Sitaraman, and Didier Merlin. ADAM-15
inhibits wound healing in human intestinal epithelial cell monolayers.
Am J Physiol Gastrointest Liver Physiol 288: G346 –G353, 2005. First
published September 9, 2004; doi:10.1152/ajpgi.00262.2004.—The
disintegrin metalloproteases (or ADAMs) are membrane-anchored
glycoproteins that have been implicated in cell-cell or cell-matrix
interactions and in proteolysis of molecules on the cell surface. The
expression and/or the pathophysiological implications of ADAMs are
not known in intestinal epithelial cells. Therefore, our aim was to
investigate the expression and the role of ADAMs in intestinal
epithelial cells. Expression of ADAMs was assessed by RT-PCR,
Western blot analysis, and immunufluorescence experiments. Wound-
healing experiments were performed by using the electric cell sub-
strate impedence sensing technology. Our results showed that
ADAMs-10, -12, and -15 mRNA are expressed in the colonic human
cell lines Caco2-BBE and HT29-Cl.19A. An ADAM-15 complemen-
tary DNA cloned from Caco2-BBE poly(A)⫹ RNA, and encompass-
ing the entire coding region, was found to be shorter and to present a
different region encoding the cytoplasmic tail compared with
ADAM-15 sequence deposited in the database. In Caco2-BBE cells
and colonic epithelial cells, ADAM-15 protein was found in the
apical, basolateral, and intracellular compartments. We also showed
that the overexpression of ADAM-15 reduced cell migration in a
wound-healing assay in Caco2-BBE monolayers. Our data show that
1) ADAM-15 is expressed in human intestinal epithelia, 2) a new
variant of ADAM-15 is expressed in a human intestinal epithelial cell
line, and 3) ADAM-15 is involved in intestinal epithelial cells wound-
healing processes. Together, these results suggest that ADAM-15 may
have important pathophysiological roles in intestinal cells.
disintegrin metalloprotease/metargidin; intestine; Caco2-BBE
A DISINTEGRIN AND METALLOPROTEASE protein (ADAM), also
called metalloprotease/disintegrin/Cys-rich (MDC), is a family
of membrane-anchored glycoproteins that present structural
similarities with the snake venom metalloproteases (36, 37).
Thirty-four ADAMs have been described in several species,
including 19 in human. ADAM proteins consist of a pro-
domain, a metalloprotease domain, a disintegrin domain, a
cysteine-rich region, an epidermal growth factor-like domain, a
transmembrane domain, and a cytoplasmic tail (5, 36, 37).
Proteins of this family have been shown to be involved in
multiple biological processes. For example, ADAMs-1, -2, and
-3 are involved in fertilization (2, 8, 10, 38), ADAM-12/
meltrin-␣ has been described as playing a role in muscle fusion
(14, 39), ADAM-17/tumor necrosis factor-␣ converting en-
Address for reprint requests and other correspondence: D. Merlin, Emory
Univ., Dept. of Medicine, Div. of Digestive Diseases, 615 Michael St., Atlanta,
GA 30322 (E-mail: dmerlin@emory.edu).
G346 0193-1857/05 $8.00 Copyright © 2005 the American Physiological Society
zyme has been shown to be involved in the processing of
pro-tumor necrosis factor-␣, L-selectin, a tumor necrosis factor
receptor, transforming growth factor-␣ (28), and ADAM-10/
Kuzbanian control cell determination and axon guidance in the
nervous system via signaling through the Notch pathway (26,
30) and through shedding of ephrins (15). The cytoplasmic tail
of ADAM proteins contains tyrosine phosphorylation sites and
proline-rich domains that allow interactions with intracellular
Downloaded from ajpgi.physiology.org on March 2, 2011
proteins involved in the cystokeleton or signal transduction
(19, 29, 31), suggesting that ADAMs could mediate intracel-
lular signaling.
Among all the ADAM proteins that have been discovered,
human ADAM-15 is the only one that has an arginine-glycine-
aspartic acid (RGD) integrin-binding motif (19), suggesting a
role of ADAM-15 in integrin binding and therefore in cell-cell
interactions. It has indeed been shown that the disintegrin
domain of ADAM-15 interacts with ␣v␤3- and ␣5␤1-integrins
in an RGD-dependent manner (25, 40) and with ␣9␤1-integrin
independently of the RGD motif (9). In addition, overexpres-
sion of ADAM-15 in the fibroblastic cell line NIH3T3 has been
found to enhance cell-cell interactions (16). ADAM-15 has
also been shown to be involved in glomerular mesangial cell
migration (22).
ADAM proteins have not been studied in intestinal epithe-
lium. ADAM-15 expression in the intestine is only poorly
documented (19), and nothing is known about its function in
the intestine. Therefore, our aim was to examine ADAM
proteins and especially ADAM-15 expression and its potential
roles in human epithelial intestinal cells.
MATERIALS AND METHODS
Cell lines and cell culture. The Caco2-BBE cell line (7, 20, 24, 33)
that differentiates at confluency into enterocyte-like cells was gener-
ously provided by Dr. M. S. Mooseker and the HT29-Cl.19A cell line
(3, 23), which differentiates at confluency into ion-transporting co-
lonic epithelial cells, was a generous gift from Dr. C. L. Laboisse.
Caco2-BBE or HT29-Cl.19A cells were grown in high-glucose
DMEM (Invitrogen, Grand Island, NY) supplemented with 14 mM
NaHCO3, 10% (vol/vol) heat-inactivated fetal bovine serum (Invitro-
gen), and 1.5 ␮g/ml plasmocin (Invivogen, San Diego, CA). Trans-
fected cells were maintained in the same medium containing 1.2
mg/ml G418 (Life Technologies, Rockville, MD). Cells were kept at
37°C, in 5% CO2, and 90% humidity. For biotinylation and confocal
experiments, confluent cells in 75 cm2 were trypsinized, plated on
porous filters (6-well or 12-well Transwell Clear, 0.4 ␮m porosity;
Costar, VWR, Suwanee, GA). Caco2-BBE cells were then cultured
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
http://www.ajpgi.org
 ADAM-15 REGULATES INTESTINAL EPITHELIAL CELL MIGRATION
for at least 10 days, and HT-29Cl.19A cells were cultured for 20 days.
For protein, membrane, or RNA extractions, Caco2-BBE cells were
plated on six-well plates (Costar) and experiments were performed at
postconfluency.
For experiments using the electric cell substrate impedance sensing
(ECIS; Applied BioPhysics, Troy, NY) system, ECIS 8W1E arrays
were used (Applied BioPhysics). Caco-2BBE cells were plated at
0.2 ⫻ 106 cells per well and experiments were performed 3 days after
seeding when cells are at confluency.
RT-PCR for ADAMs-10, -12, and -15 expression. Expression of
ADAMs-10, -12, and -15 in HT29-Cl.19A and Caco2-BBE cells
was determined by using a RT-PCR method with oligonucleo-
tide primers specific for each of these ADAMs: ADAM-10, sense:
5⬘-GCCATTACATATTCCTTCCCTTGCACAGTC-3⬘, anti-sense
5⬘-CACGAAGTTGGACATAACTTTGGATCCCCA-3⬘; ADAM-
12, sense: 5⬘-AGTGCAGAGGACTTTGAGAA-3⬘, anti-sense:
5⬘-TGCCGTTGTAGCAATAGGCT-3⬘; and ADAM-15, sense: 5⬘-
GGCTGGCAGTGTCGTCCTACCAGAGGGG-3⬘, anti-sense: 5⬘-
GGTGCACCCAGCTGCAGTTCAGCTCAGTCC-3⬘. Primers for
GAPDH were used as internal controls (GAPDH, sense: 5⬘-AC-
CACAGTCCATGCCATCAC-3⬘, anti-sense: 5⬘-TCCACCACCCT-
GTTGCTGTA-3⬘). PolyA⫹-RNA from HT29-Cl.19A or Caco2-BBE
was isolated with a Micro Fast Track kit (Invitrogen) according to the
manufacturer’s instructions. The yield of RNA from each preparation
was determined by UV spectrophotometry. Two microliters of mes-
senger RNA (⬃200 ng) were primed with oligo(dT) and reverse
transcribed with an avian myeloblastosis virus RT complementary
DNA (cDNA) Cycle Kit (Invitrogen). A dilution of the RT reaction
was used as a template for amplification by PCR. For ADAM-10 and
-15, after an initial 3-min denaturation at 94°C, 34 cycles of PCR were
performed (denaturation at 94°C for 1 min, annealing at 50°C for 1
min 30 s, and extension at 72°C for 1 min), followed by a final cycle
(denaturation at 94°C for 1 min, annealing at 50°C for 1 min 30 s, and
extension 72 °C for 10 min). For ADAM-12, PCR amplification was
performed under the same condition as described above except the
annealing temperature was 54°C instead of 50°C. For GAPDH, the
program was 2-min denaturation at 94°C, 30 cycles of denaturation at
94°C for 30 s, annealing 50°C for 2 min, and extension at 72°C for 2
min, followed by a final 10-min extension at 72°C. PCR products
were electrophoresed on ethidium bromide-stained 1% agarose gels in
Tris-acetate-EDTA (TAE).
Caco2-BBE full-length ADAM-15 complementary DNA cloning,
plasmid construction, and transfections. After RT of Caco2-BBE cells
PolyA⫹-RNA, as described above, the full-length ADAM-15 cDNA
was cloned by PCR using the following primers: sense: 5⬘-CGCT-
GTTCCGCACTTGCT-3⬘; and anti-sense: 5⬘-CCGGAGAGGTCA-
GAGGTAGA-3⬘. After an initial denaturation step at 94°C for 5 min,
PCR was carried out for 35 cycles under the following conditions:
denaturation at 94°C for 1 min, annealing at 55°C for 2 min, and
extension at 72°C for 4 min, followed by a final cycle of denaturation
at 94°C for 1 min, annealing at 55°C for 2 min, and extension at 72°C
for 10 min. The PCR product was electrophoresed on ethidium
bromide-stained 1% agarose gels in TAE. The PCR product had an
apparent size of 2.5 kb. After gel extraction, using the QIAquick Gel
Extraction Kit (Qiagen, Valencia, CA), and its ligation into a pTargeT
Mammalian Expression Vector system (Promega, Madison, WI), the
PCR product was cloned. Plasmids were then purified by using the
Maxiplasmid kit (Qiagen) and sequenced (Biosynthesis and Sequenc-
ing, Baltimore, MD). This cDNA encoding the short isoform of
ADAM-15 was then used to transfect Caco2-BBE cells. Subconfluent
Caco2-BBE cells were transfected by using Trojene Transfection
reagent (Avanti, Alabaster, AL) according to the manufacturer’s
instructions for 3– 4 h in Opti-MEM I medium (Invitrogen). Trans-
fectants were selected in medium with 1.2 mg/ml G418 (Life Tech-
nologies).
RT-PCR for ADAM-15 cytosolic domain. After RT of Caco2-BBE
cells PolyA⫹-RNA, as described above, PCR were performed with
AJP-Gastrointest Liver Physiol • VOL
G347
primers allowing the elongation of the cytosolic domain of ADAM-
15, sense: 5⬘-CTGCACCACTCAGCTAAAG-3⬘, anti-sense: 5⬘-CCG-
GAGAGGTCAGAGGTAGA-3⬘. After an initial denaturation step at
94°C for 5 min, PCR was carried out for 35 cycles under the following
conditions: denaturation at 94°C for 1 min, annealing at 55°C for 2
min, and extension at 72°C for 3 min; followed by a final cycle of
denaturation at 94°C for 1 min, annealing at 55°C for 2 min, and
extension at 72°C for 10 min. PCR products were electrophoresed on
ethidium bromide-stained 2% agarose gels in TAE.
Western blot analysis. For total protein extraction, cells were lysed
for 30 min at 4°C in RIPA buffer [150 mM NaCl, 0.5% sodium
deoxycholate, 50 mM Tris 䡠 HCl (pH 8.0), 0.1% SDS, 0.1% Nonidet
P-40] supplemented with protease inhibitors (Roche Diagnostics,
Mannheim, Germany). The homogenates were centrifuged at 13,000
g for 30 min at 4°C, and the supernatants were collected for Western
blot analysis. Protein concentrations were determined by using the
Folin assay (DC protein assay kit, Bio-Rad, Hercules, CA). Protein
extracts were mixed in tricine sample buffer (Bio-Rad), boiled for 5
min, run on a 7.5% (Bio-Rad) or an 8% (VWR) polyacrylamide gel
and then transferred to nitrocellulose membranes. Membranes were
blocked overnight at 4°C or for 1 h at room temperature with 5%
Downloaded from ajpgi.physiology.org on March 2, 2011
nonfat milk in blocking buffer and then incubated 1 h at room
temperature with a rabbit polyclonal raised against the cytoplasmic
tail of human ADAM-15 (1:1,000; R&D Systems, Minneapolis, MN).
After washing, membranes were further incubated 1 h at room
temperature with an anti-rabbit horseradish peroxidase-conjugated
antibody (1:1,000; Amersham Biosciences, Piscataway, NJ). Mem-
branes were washed again and immunoreactive proteins were detected
on films using an enhanced chemiluminescence substrate according to
the manufacturer’s instructions (Amersham Biosciences).
Cell surface biotinylation. Cells grown on filters were rinsed twice
with PBS supplemented with 0.1 mM CaCl2 and 1 mM MgCl2. Apical
or basolateral sides of the monolayers were incubated with freshly
prepared sulfosuccinimidobiotin (EZ-link sulfo-NHS-biotin; Pierce,
Rockford, IL) in PBS supplemented with 0.1 mM CaCl2 and 1 mM
MgCl2 (1 mg/ml) for 30 min at 4°C. The reaction was quenched with
50 mM NH4Cl (5 min at room temperature). Cells were then scrapped
and lysed for 30 min at 4°C in lysis buffer (immunoprecipitation kit,
protein G; Roche Diagnostics) supplemented with protease inhibitors
(Roche Diagnostics). After 30-min centrifugation (13,000 g, at 4°C),
protein concentrations were determined by using the Bio-Rad protein
assay, and the supernatants were incubated with immobilized neutra-
vidin (Pierce) overnight at 4°C to bind biotinylated proteins. After
centrifugation, the beads were washed twice in PBS (20 min at 4°C),
once in a buffer of 500 mM NaCl, 20 mM Tris pH 8.0, 0.5% Triton
X-100, and 0.2% BSA and rinsed in PBS. The beads were then boiled
5 min in tricine sample buffer (Bio-Rad), and Western blot analysis
was performed.
Membrane preparations. For membrane preparations, cells were
washed twice in PBS and scrapped in PBS. After centrifugation (400
g for 5 min), the cell pellet was resuspended and carefully homoge-
nized, with a douncer, in HEPES (5 mM) containing protease inhib-
itors, incubated for 30 min at 4°C, and then centrifuged at 13,000 g at
4°C for 30 min. The resulting pellet was suspended in PBS by
repeated passage through an 18-gauge needle. Protein concentration in
the membrane suspension and in total extracts was quantified by using
the Bio-Rad protein assay.
Confocal immunofluorescence. Caco2-BBE cells grown on filters
were washed twice with PBS pH 7.4 (Invitrogen) supplemented with
0.1 mM CaCl2 and 1 mM MgCl2 (PBS-Ca/Mg), and fixed in 4%
paraformaldehyde (Electron Microscopy Sciences, Washington, PA)
in PBS-Ca/Mg for 15 min at room temperature. After three washes in
PBS-Ca/Mg, cells were preincubated at room temperature for 30 min
in immunostaining buffer (PBS-Ca/Mg/BSA 3%/Triton X-100 0.1%)
and incubated for 40 min at room temperature with Alexa Fluor
568-conjugated phalloidin (1 unit per filter; Molecular Probes, Eu-
gene, OR) diluted in immunostaining buffer. Cells were then washed
288 • FEBRUARY 2005 • www.ajpgi.org
G348 ADAM-15 REGULATES INTESTINAL EPITHELIAL CELL MIGRATION
and exposed to a monoclonal mouse anti-ADAM-15 antibody directed
to its ectodomain (1:20; R&D Systems), in immunostaining buffer, for
1 h at room temperature. After washing, cells were incubated with a
goat anti-mouse antibody conjugated to Alexa Fluor 488 (1:200,
Molecular Probes) for 1 h at room temperature. After washing,
coverslips were mounted by using the Slowfade medium (Molecular
Probes). Microscopy was performed by using a Zeiss epifluorescence
microscope equipped with a Bio-Rad MRC600 confocal unit, com-
puter, and laser scanning microscope image analysis software (Carl
Zeiss, Jena, Germany).
Wound-healing assays. Wound-healing assays were performed with
the ECIS (Applied BioPhysics) technology (12, 13, 18). The ECIS
(model 1600R; Applied BioPhysics) was used for these experiments. The
ECIS device is based on AC impedance measurements using weak and
noninvasive AC signals as previously described in more detail (35).
Attachment and spreading of cells on the electrode surface change the
impedance in such a way that morphological information of the attached
cells can be inferred. The measurement system consists of an eight-well
culture dish (ECIS 8W1E plate), with the surface treated for cell culture.
Each well contains a small active electrode (area ⫽ 5 ⫻ 10⫺4 cm2) and
a large counter electrode (area ⫽ 0.15 cm2) deposited on the bottom of
each well. A lock-in amplifier with an internal oscillator relays to switch
among the different wells, and a personal computer controls the mea-
surement and stores the data. The entire system was obtained from
Applied Biophysics.
First, we determined the ideal frequency used to measure resistance
of confluent Caco2-BBE monolayers. To do so, we performed a
resistance measurement during a frequency scan on cells plated on
ECIS 8W1E plates (Applied BioPhysics). Resistance measurement
was the performed during a frequency scan on a naked electrode after
cell trypsinization. The ideal frequency was determined by using the
ratio of the log of resistances with cells over the log of resistances
without cells. The ideal frequency is the frequency for which the ratio
reaches a peak. For resistance measurements performed on Caco2-
BBE cells, the frequency used was 500 Hz and the voltage was 1 V.
For the wound-healing assays, confluent Caco2-BBE monolayers and
confluent Caco2-BBE that overexpressed ADAM-15 cultured on ECIS
8W1E plates were submitted to an elevated voltage pulse of 40 kHz
frequency, 4.5-V amplitude, and 30-s duration, which led to death and
detachment of cells present on the small active electrode resulting in a
wound normally healed by cells surrounding the small active electrode
that have not been submitted to the elevated voltage pulse. Wound
healing was then assessed by continuous resistance measurements for
⬃20 h. At the end of the experiments, pictures of the small active
electrodes were taken to visualize cell repair and migration.
Flow cytometry. Flow cytometry experiments were performed to
assess ADAM-15 overexpression in transfected Caco2-BBE cells.
Twenty-four hours after transfection, Caco2-BBE cells were detached
with 0.25% trypsin (Invitrogen) diluted 1:10 in PBS (without Ca2⫹
and Mg2⫹), pelleted by centrifugation, and resuspended in PBS
containing 0.5% BSA (PBS/BSA). Cells (5 ⫻ 105) were incubated
with mouse monoclonal anti-ADAM-15 (ectodomain) antibody (1:20)
in 100 ␮l PBS/BSA for 1 h at 4°C, washed twice with PBS/BSA, and
stained with a FITC-conjugated goat anti-mouse IgG (1:100; Santa
Cruz Biotechnology, Santa Cruz, CA) in 100 ␮l PBS/BSA for 1 h at
4°C. Nonspecific fluorescence was determined with FITC-conjugated
goat anti-mouse IgG. After two washes in PBS, fluorescence was
analyzed on a FACScan cytofluorometer (Becton Dickinson) with
Cell Quest software. Seven thousand cells gated on a FSC/SSC dot
plot were assayed.
RESULTS
ADAMs-10, -12, and -15 mRNAs are expressed in the
epithelial intestinal cell lines Caco2-BBE and HT29-Cl.19A.
To assess the expression of ADAMs in epithelial intestinal
cells, we performed RT-PCR experiments on epithelial intes-
AJP-Gastrointest Liver Physiol • VOL
tinal cell lines, as described in MATERIALS AND METHODS. As
shown in Fig. 1, ADAMs-10, -12, and -15 mRNA are ex-
pressed in the human colonic cell line Caco2-BBE. As ex-
pected, the size of ADAMs-10, -12 and -15 PCR products were
found to be ⬃490, 350, and 500 base pairs (bp), respectively.
The same results were found in HT29-Cl.19A cells (not
shown).
Caco2-BBE cells express two ADAM-15 isoforms. Among
all ADAMs described, human ADAM-15 has the characteristic
to possess an RGD integrin binding motif in its disintegrin
domain (19). We therefore focused on ADAM-15, and the
full-length cDNA encoding ADAM-15 in Caco2-BBE cells
was cloned and sequenced as described in MATERIALS AND
METHODS. Our results showed that the cloned ADAM-15 cDNA
presents a 70-bp deletion in the region encoding the cytoplas-
mic tail (from 2,208 to 2,277 bp), in ADAM-15 cDNA (Gen-
Bank accession no. AY518542) compared with published
ADAM-15 cDNA (GenBank accession nos. 2208386A,
AAC50404, AAM44189, AAP88766, Q13444, NP_003806,
Downloaded from ajpgi.physiology.org on March 2, 2011
and BC014566) (Fig. 2A). This 70-bp deletion results in a
change in the open reading frame of ADAM-15 mRNA,
leading to the generation of a stop codon (UAG) at 2,317 bp
instead of the stop codon UGA, located at 2,443 bp (Fig. 2A).
As represented in Fig. 2B, the ADAM-15 protein generated
from this mRNA is therefore shorter (772 amino acids instead
of 814), and its cytoplasmic tail differs after amino acid 735.
To further investigate the expression of this mRNA in Caco2-
BBE cells, we performed RT-PCR experiments using primers
allowing the elongation of the cytoplasmic tail of ADAM-15,
as described in the MATERIALS AND METHODS. Two PCR products
were found: one corresponding to the full-length cytoplasmic
tail (406 bp, long isoform) and one corresponding to the 70-bp
deletion-containing cytoplasmic tail (336 bp, short isoform)
(Fig. 2C). Figure 2D represents the amino acids sequence of
ADAM-15 cytoplasmic tail for the long (upper sequence) and
for the short (lower sequence) isoforms. The cytoplasmic tail
of ADAM-15 presents some motifs suggesting that ADAM-15
might transduce intracellular signals. Potential proline-rich
domains (19, 31) are represented in green (Fig. 2D). Tyrosines
represented in blue (Y715 and Y735) have been described as
phosphorylated (29) and serine and threonine represented in
red are potential phosphorylation sites according to the Net-
Phos 2.0 Server (Fig. 2D). The cytoplasmic tail of the short
isoform of ADAM-15 is 42 amino acids shorter; it comprises
61 amino acids, whereas the cytoplasmic tail of the long
Fig. 1. A disintegrin and metalloproteases (ADAMs)-10, -12, and -15 mRNA
are expressed in human intestinal cell lines. Samples isolated from the human
epithelial colonic HT29-Cl19A cell line were subjected to RT-PCR using
GAPDH-, ADAM-15-, ADAM-12-, and ADAM-10-specific primers as de-
scribed in MATERIALS AND METHODS. The RT-PCR products were analyzed by
agarose gel electrophoresis. Numbers on the right are the molecular size of
standards in base pairs.
288 • FEBRUARY 2005 • www.ajpgi.org
 ADAM-15 REGULATES INTESTINAL EPITHELIAL CELL MIGRATION G349

Downloaded from ajpgi.physiology.org on March 2, 2011

Fig. 2. Caco2-BBE cells express a long and a short isoform of ADAM-15. Cloning and sequencing of the full-length ADAM-15 cDNA from Caco2-BBE cells,
as described in MATERIALS AND METHODS, showed that Caco2-BBE cells express a new variant of ADAM-15 mRNA with a 70-bp deletion (A) leading to the
translation of a shorter protein with a cytoplasmic (Cys) tail that differs after amino acid 735 (B). C: RT-PCR experiments performed on Caco2-BBE cells, using
primers encompassing the ADAM-15 cytoplasmic tail, showed that Caco2-BBE cells express two isoforms of ADAM-15. Numbers on the left are the molecular
size of standards. D: the cytoplasmic domain of the long isoform ADAM-15 contains potentials of proline-rich sequences (green), potential serine and threonine
phosphorylation sites (red), and two tyrosines (Y715 and Y735) described as undergoing phosphorylation (blue). In the cytoplasmic tail of the short isoform,
Y715 and Y735 are conserved (blue), and there are potential serine and threonine phosphorylation sites (red). Amino acids in italic and underlined represent the
part of the short cytoplasmic domain that differs from the long cytoplasmic tail.

isoform comprises 103 amino acids (Fig. 2D). This shorter cell membrane as well as in the intracellular compartment.
cytoplasmic tail possesses tyrosines 715 and 735, and, accord- Within the membrane, ADAM-15 was found to be expressed in
ing to the NetPhos 2.0 Server, it contains two serines and one both apical and basolateral compartments, although the stain-
threonine that are potential phosphorylation sites. However, ing was more intense in the apical compartment (Fig. 4A).
this short cytoplasmic tail lacks all the predicted proline-rich
domains found in the long cytoplasmic tail, and the environ-
ment of tyrosine 735 is not the same (Fig. 2D), suggesting that
this tyrosine might not be phosphorylated in the short isoform.
This suggests that the short and long isoforms of ADAM-15
might transduce different intracellular signals.
ADAM-15 protein is expressed in human intestinal epithelial
cells. ADAM-15 expression in the intestine was further exam-
ined at the protein level. Immunoblot experiments performed
on human biopsy whole cell extracts showed that both pro form
(110 kDa) and mature form (85 kDa) of ADAM-15 protein are
expressed in human small intestine (Fig. 3A). Surprisingly,
only one band of ⬃90 kDa was revealed in human colon
extracts (Fig. 3A). The enterocyte-like Caco2-BBE cell line
was then used as a model system to assess ADAM-15 expres-
sion in human intestinal epithelial cells. Immunoblot experi-
ments performed on whole cell extracts as well as on mem-
brane preparations from Caco2-BBE cells showed that both pro
form and mature form of ADAM-15 are present in the intra- Fig. 3. ADAM-15 protein is expressed in human normal small intestine and
cellular compartment as well as at the cell surface membrane colon, and in the human enterocyte-like Caco2-BBE cell line. Immunoblot anal-
(Fig. 3B). ysis of ADAM-15 expression was performed as described in MATERIALS AND
METHODS from lysates of human small intestine (30 ␮g protein loaded) and colon
To assess the localization of ADAM-15 in Caco2-BBE cells,
biopsies (80 ␮g protein loaded) (A) and from whole cell extract (50 ␮g protein
we performed immunofluorescence staining using a mouse loaded) and membrane preparation (25 ␮g protein loaded) of Caco2-BBE cells,
anti-ADAM-15 antibody raised against the ectodomain. As using the polyclonal rabbit anti-ADAM-15 cytoplasmic tail antibodies (B). Num-
shown in Fig. 4A, ADAM-15 was found to be expressed on the bers on the right are the molecular size of standards in thousands.

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G350 ADAM-15 REGULATES INTESTINAL EPITHELIAL CELL MIGRATION

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Fig. 4. ADAM-15 is expressed in both apical and basolateral membranes and in the intracellular compartment in Caco2-BBE cells. A: confocal microscopic
localization of ADAM-15 (green) in Caco2-BBE cell monolayers also stained with phalloidin (actin, red). a, horizontal (xy) section near the apical plasma
membrane; b, horizontal (xy) section near the basolateral plasma membrane and vertical (zy) section of polarized Caco2-BBE monolayers stained with the
anti-ADAM-15 ectodomain and phalloidin. CTRL, control. B: filter-grown Caco2-BBE monolayers were subjected to domain-specific biotinylation [apical
(Apic.), basolateral (Basol.)] for 30 min followed by Western blot analysis of biotinylated proteins. Biotinylated proteins were subjected to 7.5% SDS-PAGE
followed by transfer to nitrocellulose membrane. The blot was immunostained with rabbit anti-ADAM-15 antibody (raised against the cytoplasmic domain).

These data were confirmed by cell surface biotinylation exper-
iments, which showed that although both forms of ADAM-15
are expressed in both apical and basolateral compartments, the
mature form of ADAM-15 is expressed in a much higher level
in the apical compartment (Fig. 4B).
ADAM-15 regulates intestinal epithelial cell wound-healing.
ADAM-15 has been described as having a protease activity
resulting in type IV collagen and gelatin degradation (22) and
in CD23 shedding (11). Because cell migration involves pro-
tease activity and ADAM-15 is strongly expressed in human
epithelial intestinal cells, we hypothesized that ADAM-15
might be involved in epithelial cell migration along intestinal
crypts. To test this hypothesis, we performed a wound-healing
assay using the ECIS technology (12, 13, 18) and using the
Caco2-BBE cell line as a model system. Experiments were
conducted as follows. Cells were seeded on plates containing
gold-film electrodes through which elevated field pulses were
applied and that also measure cell resistance. We first deter-
mined the ideal frequency used to measure resistance of
Caco2-BBE cells. We performed resistance measurements dur-
ing a frequency scan on ECIS 8W1E plates, first in the
presence of confluent cells, and then without cells on naked
electrodes as described in the MATERIALS AND METHODS. Figure
5A represents the profiles of resistances obtained for different
frequencies in eight wells. The upper set of curves represents
the measurements taken in the presence of confluent cells and
the lower set of curves represents the measurements taken in
the same wells after cell trypsinization. It is then possible to
determine the ideal frequency as it is represented in Fig. 5B,
using the ratio of the log of resistances with cells over the log
of resistances without cells. The ideal frequency is the fre-
AJP-Gastrointest Liver Physiol • VOL
quency for which the ratio reaches a peak. For Caco2-BBE
cells, the ratio peak was found for a log [frequency (Hz)] of
2.7, corresponding to a frequency of 500 Hz (Fig. 6B). All the
resistance measurements performed on Caco2-BBE cells, us-
ing the ECIS technology, were therefore performed at a fre-
quency of 500 Hz.
To investigate the role of ADAM-15 in wound-healing of
human epithelial intestinal cells, we performed wound-healing
experiments with Caco2-BBE cells transfected either with the
short isoform of ADAM-15 or with the vector alone. To assess
ADAM-15 overexpression in transfected cells, we performed
flow cytometry experiments 24 h after transfection. Figure 6A,
inset, shows that as early as 24 h after transfection, cells
transfected with ADAM-15 (Caco2-BBE/ADAM-15) start ex-
pressing a higher level of ADAM-15 compared with cells
transfected with the vector only (Caco2-BBE/Vect).
For the wound-healing assays, Caco2-BBE/Vect and Caco2-
BBE/ADAM-15 cells cultured on ECIS 8W1E plates were
submitted to an elevated voltage pulse of 40 kHz frequency,
4.5 V amplitude, and 30-s duration, and resistance measure-
ments were then performed. At the end of the experiments, i.e.,
19 h after the elevated voltage pulse application, cells on
electrodes were observed under a microscope and pictures
were taken. As shown in Fig. 6A, the application of the
high-field pulse led to a drastic drop of cell resistance. After the
wound, the resistances of Caco2-BBE/ADAM-15 cells did not
increase to reach the resistance values of control, nonwounded
cells (Fig. 6A). By contrast, the resistances of Caco2-BBE/Vect
cells increased over time and reached the resistance values of
control, nonwounded cells (Fig. 6A). These results were in
correlation with the observations made at the end of the
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 ADAM-15 REGULATES INTESTINAL EPITHELIAL CELL MIGRATION
Fig. 5. Determination of the ideal frequency for resistance measurements on
Caco2-BBE cells with the electric cell substrate impedance sensing (ECIS)
technology. A: resistances of confluent Caco2-BBE cells in ECIS plates (top
curves) or of naked electrodes, after cell trypsinization (bottom curves) were
measured at different frequencies. Graph represents the log of the resistance
measured in ohms and the log of the frequency applied for the measurement in
hertz. Each color represents one well, with or without cells. B: frequency for
which the ratio (log resistance with cells/log resistance without cells) reaches
a peak is the suitable frequency for resistance measurement.
experiments that Caco2-BBE/Vect cells completely healed the
wound, whereas Caco2-BBE/ADAM-15 cells were unable to
heal the wound (Fig. 6B).
DISCUSSION
The role of the ADAM molecules in tissue functions is
poorly understood. These molecules have separate domains
that might have multiple functions, such as membrane fusion,
cytokine and growth factor shedding, and cell adhesion and
migration (31).
In the present study, we demonstrate for the first time that
some ADAMs including ADAM-15 are expressed in human
intestinal epithelial cells. Interestingly, human ADAM-15 is
the only known ADAM to contain an RGD peptide in the
predicted integrin-binding loop of the desintegrin domain (19).
AJP-Gastrointest Liver Physiol • VOL
G351
Another interesting function of ADAM-15 involves the cyto-
plasmic domain, because it has been reported that it possesses
motifs likely to be involved in signal transduction (19, 29, 31).
Our results have shown that Caco2-BBE cells express two
isoforms of ADAM-15 protein: one long isoform, correspond-
ing to the isoform deposited on the DNA database, and one
short isoform presenting a 70-bp deletion resulting in a shorter
and partly different cytoplasmic tail. It has been shown that the
cytoplasmic tail of ADAM-15 binds with two SH3-containing
proteins, endophilin I and SH3PX1 (17). Poghosyan et al. (29)
have shown that two tyrosines (Y715 and Y735) within the
cytoplasmic tail of ADAM-15 are phosphorylated and can bind
with proteins of the Src family protein-tyrosine kinases. We
have shown that the deletion leads to a different cytoplasmic
tail that does not contain the predicted proline-rich domains
present in the long cytoplasmic tail and for which the environ-
ment of tyrosine 735 is changed. It is interesting to speculate
that each of the two different extensions of the ADAM-15
cytoplasmic tail represents a modular unit designed to interact
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with unidentified cytoplasmic or membrane proteins. It is
likely that the different tails might interact differently with
cytoskeletal elements or integrins or transduce different signal
cascades to mediate different cell-specific functions. These
results are in agreement with previous studies (32) showing
that murine T lymphocytes and monocytes lines express three
isoforms of ADAM-15. These isoforms differ in their cyto-
plasmic domain and present differential interaction among
their cytoplasmic domains and Src family protein-tyrosine
kinases (32).
Our results demonstrate that ADAM-15 protein is expressed in
Caco2-BBE monolayers and in human epithelial intestinal cells in
the intracellular compartment, as well as at the cell surface
membrane. On the cell surface of Caco2-BBE monolayers, both
pro-ADAM-15 and processed ADAM-15 can be detected, al-
though both forms apparently only represent a small amount of
the total ADAM-15 in the cell. However, the precursor found on
the cell surface could be processed at a later stage after endocy-
tosis (21). It has been suggested that ADAM-15 processing may
require endocytosis from the cell surface (21). Interestingly, the
major form of ADAM-15 detected by Western blot analysis found
in colonic tissue seems to be different than the one found in the
small intestine or in Caco2-BBE cells and might be explained by
different degrees of glycosylation. Human ADAM-15 is the only
ADAM with an RGD motif within the putative integrin-binding
sites of desintegrin domains (19). The presence of the RGD
domain is suggested to play a role in cell-cell adhesion (19). In
addition, it is known that the RGD domain interacts with ␤2-
integrins that are expressed in immune cells (27). Presence of a
transmembrane protein that contains an RGD domain in intestinal
epithelial cells may have some pathophysiological relevance. For
example, during intestinal inflammation, leukocyte ␤2-integrins
CD11b/CD18 and CD11c/CD18 are known to bind to the baso-
lateral aspect of intestinal epithelial cells (27). The expression of
the ␤2-counter receptor in intestinal epithelial cells might help
neutrophils to bind to the epithelium. This scenario is possible,
because we have shown that ADAM-15 protein is expressed in
the basolateral membrane of Caco2-BBE monolayers and in
human intestinal epithelial cells. However, on the basis of our in
vitro and in vivo results, ADAM-15 is likely to be expressed to the
apical aspect of intestinal epithelial. It is conceivable that the
metalloprotease domain may play an important proteolytic activ-
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G352 ADAM-15 REGULATES INTESTINAL EPITHELIAL CELL MIGRATION

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Fig. 6. Overexpression of ADAM-15 impairs Caco2-BBE cell wound healing. After transfection of Caco2-BBE cells with the short isoform of ADAM-15
(Caco2-BBE/ADAM-15) or with the vector alone (Caco2-BBE/Vect), ADAM-15 expression was assessed by flow cytometry (A, inset). Confluent Caco2-BBE/
ADAM-15 and Caco2-BBE/Vect cells, seeded in ECIS plates, were submitted (wounded) or not (ctl) to an elevated voltage pulse of 40 kHz frequency, 4.5 V
amplitude, and 30-s duration. The wound was then allowed to heal from cells that did not undergo the elevated voltage pulse and that surround the small active
electrode. A: resistance was measured before and after the elevated voltage pulse application as described in the MATERIALS AND METHODS. The measurements
were stopped ⬃19 h after the wound. B: at the end of the experiments, cells were washed twice in PBS, and pictures were taken of cells on the electrodes. Arrows
indicate the direction of cell repair and migration.

ity against the lumen content. This function is supported by the
fact that the mature, proteolytically active, ADAM-15 is the
dominant form expressed in the apical plasma membrane of
Caco2-BBE monolayers.
Colonic epithelial cells undergo cell cycle arrest, lineage-
specific differentiation, and apoptosis as they migrate along the
crypt axis to reach the luminal surface. The increase in extra-
cellular matrix (ECM)-associated genes suggests that increased
deposition of an ECM may be a function of differentiated
colonic epithelial cells. Indeed, interaction of epithelial cells
with ECM components is known to modulate differentiation
(4). In the present study, we observed that ADAM-15 is
expressed in human intestinal epithelial cells, suggesting that it
may play a role in intestinal epithelial cell migration and
differentiation. Because ADAM-15 possesses a protease activ-
ity (11, 22) and because cell migration processes involve
protease activity, we investigated the role of ADAM-15 in
intestinal epithelial cells migration. To examine this hypothe-
sis, wound-healing experiments were performed by using the
ECIS technology. The ECIS instrument allows performing
automated and accurate wound-healing assays. The ECIS
wound is very well defined, because it includes only cells
grown on the 250-␮m diameter electrode, i.e., an area of ⬃5 ⫻
10⫺4 cm2. In our system, overexpression of ADAM-15 in
Caco2-BBE cells resulted in the inhibition of wound-healing
processes. These results are consistent with the work of Herren
et al. (16) who showed that ADAM-15 overexpression in the
fibroblastic cell line NIH3T3 inhibited cell migration. The
inhibition of wound repair we observed in Caco2-BBE over-
expressing ADAM-15 could be due to an enhancement of
AJP-Gastrointest Liver Physiol • VOL
cell-cell interactions as suggested by the work of Herren et al.
(16) on NIH3T3 cells overexpressing ADAM-15. As has
already been suggested (1), ADAM-15 could inhibit cell mi-
gration by interacting with integrins and therefore balancing
interactions between integrins and matrix.
An impairment of the integrity of the mucosal epithelial barrier
is observed in the course of various intestinal disorders including
inflammatory bowel diseases, celiac disease, intestinal infections,
and various other diseases. The integrity of the intestinal mucosal
surface barrier is reestablished by resealing of the surface epithe-
lium through epithelial cell migration, proliferation, and differen-
tiation. Our results showing that ADAM-15 plays a role in wound
healing therefore suggest that ADAM-15 might be involved in
intestinal pathologies, such as inflammatory bowel diseases. In
addition, the fact that ADAM-15 overexpression inhibits wound
healing processes suggests that ADAM-15 could prevent inappro-
priate cell migration and therefore metastasis formation. For that
matter, in vivo studies have shown that the recombinant dis-
integrin domain of ADAM-15 possesses an anti-metastatic activ-
ity (34).
Further studies about the status and the function of
ADAM-15 in vivo are needed to confirm the validity of these
in vitro findings. ADAM-15 expression has already been
shown to be dysregulated in some pathologies. For example,
ADAM-15 expression has been shown to be upregulated in
atherosclerosis (1) as well as in osteoarthritic cartilage (6).
In this report, we show for the first time that 1) ADAM-15
protein is expressed in both native human intestinal epithelium
as well as in human intestinal epithelial cell lines; 2) a human
intestinal epithelial cell line expresses two isoforms of
288 • FEBRUARY 2005 • www.ajpgi.org
 ADAM-15 REGULATES INTESTINAL EPITHELIAL CELL MIGRATION
ADAM-15 that differ in their cytoplasmic tail, suggesting
differential signal transduction and therefore different roles;
and 3) ADAM-15 regulates wound-healing processes in human
intestinal epithelial cells.
GRANTS
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases under a center grant (R24-DK-064399) and 1R01-DK-
061941– 01A1 (to D. Merlin), KO8-DK-02802 and 1R03-DK-064644 – 01 (to
S. V. Sitaraman), and a Research Fellowship Award from the Crohn’s and
Colitis Foundation of America (to L. Charrier).
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