CXCR4 regulates migration of lung alveolar epithelial cells through activation of Rac1 and matrix metalloproteinase-2.

Abstract

Restoration of the epithelial barrier following acute lung injury is critical for recovery of lung homeostasis. After injury, alveolar type II epithelial (ATII) cells spread and migrate to cover the denuded surface and, eventually, proliferate and differentiate into type I cells. The chemokine CXCL12, also known as stromal cell-derived factor 1α, has well-recognized roles in organogenesis, hematopoiesis, and immune responses through its binding to the chemokine receptor CXCR4. While CXCL12/CXCR4 signaling is known to be important in immune cell migration, the role of this chemokine-receptor interaction has not been studied in alveolar epithelial repair mechanisms. In this study, we demonstrated that secretion of CXCL12 was increased in the bronchoalveolar lavage of rats ventilated with an injurious tidal volume (25 ml/kg). We also found that CXCL12 secretion was increased by primary rat ATII cells and a mouse alveolar epithelial (MLE12) cell line following scratch wounding and that both types of cells express CXCR4. CXCL12 significantly increased ATII cell migration in a scratch-wound assay. When we treated cells with a specific antagonist for CXCR4, AMD-3100, cell migration was significantly inhibited. Knockdown of CXCR4 by short hairpin RNA (shRNA) caused decreased cell migration compared with cells expressing a nonspecific shRNA. Treatment with AMD-3100 decreased matrix metalloproteinase-14 expression, increased tissue inhibitor of metalloproteinase-3 expression, decreased matrix metalloproteinase-2 activity, and prevented CXCL12-induced Rac1 activation. Similar results were obtained with shRNA knockdown of CXCR4. These findings may help identify a therapeutic target for augmenting epithelial repair following acute lung injury.

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doi: 10.1152/ajplung.00321.2011
302:L846-L856, 2012. First published 17 February 2012;
Am J Physiol Lung Cell Mol Physiol
M. Waters
Manik C. Ghosh, Patrudu S. Makena, Vijay Gorantla, Scott E. Sinclair and Christopher
metalloproteinase-2
cells through activation of Rac1 and matrix
CXCR4 regulates migration of lung alveolar epithelial
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CXCR4 regulates migration of lung alveolar epithelial cells through activation
of Rac1 and matrix metalloproteinase-2
Manik C. Ghosh,1Patrudu S. Makena,2Vijay Gorantla,2Scott E. Sinclair,1,2
and Christopher M. Waters1,2,3
Departments of1Physiology,2Medicine, and3Biomedical Engineering and Imaging, University of Tennessee Health Science
Center, Memphis, Tennessee
Submitted 5 October 2011; accepted in final form 13 February 2012
Ghosh MC, Makena PS, Gorantla V, Sinclair SE, Waters CM.
CXCR4 regulates migration of lung alveolar epithelial cells through
activation of Rac1 and matrix metalloproteinase-2. Am J Physiol Lung
Cell Mol Physiol 302: L846–L856, 2012. First published February 17,
2012; doi:10.1152/ajplung.00321.2011.—Restoration of the epithelial
barrier following acute lung injury is critical for recovery of lung
homeostasis. After injury, alveolar type II epithelial (ATII) cells
spread and migrate to cover the denuded surface and, eventually,
proliferate and differentiate into type I cells. The chemokine
CXCL12, also known as stromal cell-derived factor 1?, has well-
recognized roles in organogenesis, hematopoiesis, and immune re-
sponses through its binding to the chemokine receptor CXCR4. While
CXCL12/CXCR4 signaling is known to be important in immune cell
migration, the role of this chemokine-receptor interaction has not been
studied in alveolar epithelial repair mechanisms. In this study, we
demonstrated that secretion of CXCL12 was increased in the bron-
choalveolar lavage of rats ventilated with an injurious tidal volume
(25 ml/kg). We also found that CXCL12 secretion was increased by
primary rat ATII cells and a mouse alveolar epithelial (MLE12) cell
line following scratch wounding and that both types of cells express
CXCR4. CXCL12 significantly increased ATII cell migration in a
scratch-wound assay. When we treated cells with a specific antagonist
for CXCR4, AMD-3100, cell migration was significantly inhibited.
Knockdown of CXCR4 by short hairpin RNA (shRNA) caused
decreased cell migration compared with cells expressing a nonspecific
shRNA. Treatment with AMD-3100 decreased matrix metalloprotei-
nase-14 expression, increased tissue inhibitor of metalloproteinase-3
expression, decreased matrix metalloproteinase-2 activity, and pre-
vented CXCL12-induced Rac1 activation. Similar results were ob-
tained with shRNA knockdown of CXCR4. These findings may help
identify a therapeutic target for augmenting epithelial repair following
acute lung injury.
acute respiratory distress syndrome; epithelial repair; acute lung
injury
ACUTE RESPIRATORY DISTRESS syndrome is a severe form of lung
injury with high mortality and morbidity (10, 58, 62). A
prominent feature of acute respiratory distress syndrome is
injury to the endothelial and epithelial barrier. The specific
mechanisms of repair and the restoration of lung barrier func-
tion are incompletely understood. Migration of local epithelial
stem cells and recruitment of other progenitor cells to repair the
damaged tissue are crucial for recovery of the injured lung (2,
48). These cells are stimulated and recruited by growth factors,
cytokines, and chemokines released at the site of injury (14).
Alveolar type II (ATII) cells are thought to be the predominant
local stem cell that repairs the alveolar epithelial surface (54).
Accumulated evidence indicates that ATII cells express growth
factor and cytokine receptors that are involved in repair,
including receptors for hepatocyte growth factor, keratinocyte
growth factor, EGF, FGF-10, IL-2, and IL-1? (24, 33, 34, 43,
44, 56).
Chemokines are low-molecular-weight proteins that have
been shown to involved in cell migration, proliferation, differ-
entiation, and inflammation in asthma, chronic obstructive
pulmonary disease, and acute lung injury (7, 8, 12, 28, 37, 42).
However, their role in ATII cell-mediated repair has not been
extensively studied. Chemokines have also been shown to
control the expression of adhesion receptors on cells and,
thereby, influence interactions with the extracellular matrix
(ECM) (47). ATII cells have been shown to express the
chemokine receptors CCR2, CCR3, CXCR2, and CXCR3 (7,
12, 46, 57), and stimulation of CCR2 by its ligand monocyte
chemoattractant protein 1 has been shown to stimulate repair of
wounded cell monolayers. Mice lacking the CXCR4 gene or
the gene for its ligand CXCL12 (stromal cell-derived factor
1?) do not survive (40, 55). Expression of CXCR4 has been
shown in alveolar epithelial cells (39), but the role of CXCR4
in epithelial repair has not been investigated.
In the present study, we report that primary cultures of rat
ATII cells and a mouse lung epithelial (MLE12) cell line
express CXCR4, and this receptor is activated by binding with
its ligand CXCL12. Our data indicate a novel role for CXCR4
in the repair of wounded ATII cells involving Rac1, matrix
metalloproteinase (MMP)-14, and MMP-2.
MATERIALS AND METHODS
Reagents. DMEM-penicillin-streptomycin-trypsin-EDTA solution
and PBS were purchased from GIBCO Life Technologies (Grand
Island, NY); FBS from Hyclone (Logan, UT); HEPES, KCl, MgCl2,
Triton X-100, sodium vanadate, PMSF, aprotinin, and leupeptin from
Sigma (St. Louis, MO); Tween 20 from Bio-Rad (Hercules, CA);
rhodamine-phalloidin and DRAQ-5 (a nuclear stain) from Molecular
Probes (Eugene, OR); AMD-3100 [a highly specific antagonist of
CXCR4 (20)] and the Rac1 inhibitor W56 from Sigma Aldrich (St.
Louis, MO); the Rac1 and CDC42 activation assay kit from Millipore
(Billerica, MA); gradient gels (4–12%) for Western blot and 10%
gelatin gel, renaturation buffer, and developing buffer for zymography
from Invitrogen (Carlsbad, CA); antibodies of CXCR4 (catalog nos.
ab13854, sc-9046, and LS-C250) from Abcam (Cambridge, MA),
Santa Cruz Biotechnology (Santa Cruz, CA), and LS Biosciences
(Seattle, WA), respectively; CXCR4 blocking antibody, control iso-
type IgG for CXCR4, and an antibody for MMP-2 from Santa Cruz
Biotechnology; and monoclonal antibodies for MMPs specific for
MMP-1, MMP-3, MMP-9, MMP-11, MMP-12, MMP-13, MMP-14,
and MMP-21 from Epitomics (Burlingame, CA).
Cell culture. Primary rat ATII cells were isolated according to the
methods described previously (16, 19). The animal use protocol was
Address for reprint requests and other correspondence: C. M. Waters, Univ.
of Tennessee Health Science Center, 894 Union Ave., 426 Nash, Memphis, TN
38163 (e-mail: cwaters2@uthsc.edu).
Am J Physiol Lung Cell Mol Physiol 302: L846–L856, 2012.
First published February 17, 2012; doi:10.1152/ajplung.00321.2011.
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approved by the Institutional Animal Care and Use Committee at the
University of Tennessee Health Science Center. Briefly, ATII cells
were isolated from male Sprague-Dawley rats by elastase digestion
and differential adherence on IgG-coated dishes. ATII cells were
identified using Nile Red (Sigma) staining of lamellar bodies, and
?90% of the cells were Nile Red-positive on day 2. We used plastic
six-well plates or chamber slides that were coated with rat lung
fibroblast (RLF) matrix deposited by RLF-6 cells (American Type
Culture Collection) for wound-healing assays with ATII cells (16).
Freshly isolated cells were seeded to confluence at 3.5 ? 106cells/
well in ATII culture medium (DMEM with 10% FBS, 4 mM glu-
tamine, 1% penicillin-streptomycin, and 0.25 ?M amphotericin B),
and experiments were performed on day 2 after isolation. MLE12
cells, an alveolar epithelial cell line established from pulmonary
tumors in transgenic mice (30), were obtained from the American
Type Culture Collection and cultured using medium containing
DMEM with 10% FBS, 4 mM glutamine, and 1% penicillin-strepto-
mycin.
Measurement of CXCL12. CXCL12 in the conditioned media of
ATII and MLE12 cells and in the bronchoalveolar lavage fluid (BAL)
from rats was measured using an ELISA according to the manufac-
turer’s protocol (NovaTeinBio, Cambridge, MA). Samples were col-
lected from control cells or cells subjected to multiple scratch wound-
ing 24 h earlier. Multiple scratch wounds were made as previously
described (18). BAL was collected from control rats or from rats
following 4 h of mechanical ventilation with an injurious tidal volume
of 25 ml/kg body wt using the FlexiVent (SCIREQ, Montreal, PQ,
Canada) with room air, as previously described (35).
Development of the CXCR4-shRNA cell line. A stable cell line with
decreased expression of CXCR4 was developed in MLE12 cells using
a short-hairpin RNA (shRNA; catalog no. SC-35422-V, Santa Cruz
Biotechnology) according to the manufacturer’s protocol. A cell line
with control shRNA was developed in the same cell line using a
scrambled vector (catalog no. SC-108080, Santa Cruz Biotechnol-
ogy). The antibiotic puromycin was used in the media to select the
positive clone. The knockdown status of CXCR4 was assessed by
Western blotting for CXCR4 protein expression.
Zymography. MMP-2 activity was determined using gelatin zy-
mography (45). Cells were seeded at equal densities and grown to
confluence. An equivalent volume of medium (2 ml) was conditioned
by the cells for 24 h. Cell lysates and conditioned media containing
equal amounts of protein were run in native condition (nondenatured)
in 10% gelatin gels. Gels were incubated first in renaturation buffer
and then in developing buffer according to the manufacturer’s proto-
col (Invitrogen). The final zymogram was developed by exposure of
the gel to staining solution (1% Coomassie blue in 40% methanol,
10% acetic acid, and 50% water) and then to destaining solution
without Coomassie blue (40% methanol, 10% acetic acid, and 50%
water). A white band on the gel indicated the activated MMP-2 that
cleaved the gelatin substrate on the gel. MMP-2 appeared as a
?66-kDa band. The specificity of MMP-2 was confirmed by immu-
noblot by screening for MMPs using specific antibodies.
Small interfering RNA knockdown of MMP-14. MMP-14 was
transiently knocked down in primary rat ATII cells using small
interfering RNA (siRNA; Dharmacon). siRNA was transfected into
semiconfluent ATII cells on day 1 after isolation using an siRNA
transfection kit (Santa Cruz Biotechnology), and experiments were
begun on day 3 after isolation.
Wound-healing assay. Cell migration was measured according to
our previous methods (15). Confluent monolayers of ATII, MLE12, or
CXCR4-shRNA cells were wounded by scraping a pipette tip across
the monolayer to produce initial 1,000- to 1,200-?m wounds. Images
were collected with a Cool Snap charge-coupled device camera
(Roper Scientific, Trenton, NJ) mounted on an Eclipse TE300 in-
verted microscope with a ?4 objective (Nikon, Melville, NY). Images
were obtained at the initial time of wounding and then 16–24 h after
wounding. Metamorph software was used to record the coordinates
for each wound location using a computer-controlled stage, so that the
same location was used for postwounding measurement, and data were
analyzed by Metamorph imaging software. The mean wound width at 24 h
was calculated and normalized to the original wound width as a
percentage. All results are from at least three independent wells from
at least two separate experiments (n ? 6).
SDS-PAGE. Protein concentration was determined by the Bradford
method (9). Equal amounts of protein were resolved by 4–12%
SDS-PAGE and electrophoretically transferred onto PVDF mem-
branes. Membranes were blocked for 1 h in 5% nonfat milk (Bio-Rad)
in Tris-buffered saline containing 0.01% Tween 20 (TBST) and
incubated overnight at 4°C with the appropriate primary antibody.
Membranes were washed with TBST and then incubated with the
secondary antibody for 1 h at room temperature. Finally, blots were
developed on X-ray film using the enhanced chemiluminescence
method (Amersham Biosciences, Piscataway, NJ).
Activated Rac1 assay. Rac1 activity assays were performed
according to the manufacturer’s instructions (Millipore). ATII,
CXCR4-shRNA, and scrambled shRNA cells were pretreated with
or without AMD-3100 (2 ?g/ml) for 1 h. Pretreated cells were
exposed to rat or mouse CXCL12 (100 ng/ml) for 2 min. Ice-cold
PBS was added to the wells, which were washed twice. Finally,
cells were harvested by addition of 100 ?l of ice-cold lysis buffer
in each well (supplied in the kit) mixed with a cocktail of protease
inhibitors (10 ?l/ml; catalog no. 78425, Thermo Scientific, Rock-
ford, IL). Cell lysates were centrifuged at 14,000 g at 4°C for 10
min, and an equal amount of protein in an equal volume of lysis
buffer (500 ?l) was incubated with p21-activated kinase, which
was chemically coupled with agarose beads at 4°C for 1 h. The
beads were pelleted by brief centrifugation (10,000 g at 4°C for 10
s) and washed three times with the lysis buffer. GTP-bound Rac1
was solubilized in Laemmli sample buffer and separated using a
4–12% Bis-Tris gel for Western blotting. Anti-Rac1 (1:500 dilu-
tion) and horseradish peroxidase-conjugated anti-mouse secondary
(1:5,000 dilution; Cell Signaling Technology, Boston, MA) anti-
bodies were used to probe the Rac1 in Western blots.
Confocal microscopy. Expression of CXCR4 in ATII and MLE12
cells was assessed by confocal microscopy. ATII or MLE12 cells
were grown to confluency on precleaned coverslips and then fixed
Fig. 1. CXCL12 secretion was increased following injury in rats and in
cultured cells. CXCL12 was measured by ELISA in the conditioned media of
rat alveolar type II (ATII) or mouse lung epithelial (MLE12) cells 24 h after
multiple scratch wounds were applied to the confluent cells. Control cells were
unwounded. CXCL12 was also measured in bronchoalveolar lavage fluid
(BAL) from control rats and rats ventilated with a tidal volume of 25 ml/kg.
Values are means ? SE (n ? 3). *P ? 0.05 vs. control.
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with 4% paraformaldehyde. Cells were permeabilized with 0.1%
Triton X-100 and then blocked with 2% BSA. CXCR4 primary
antibody was incubated at 1:100 dilution overnight at 4°C. The
secondary antibody labeled with Alexa Fluor 488 (Molecular
Probes) was used at 1:500 dilution at room temperature for 30 min.
The nucleus was stained with DRAQ5 (Molecular Probes) at
1:1,000 dilution for 15 min at room temperature. Images were
obtained using a confocal microscope (Olympus) with ?20 and
?40 objectives. For examination of F-actin distribution in migrat-
ing cells, control and CXCR4-shRNA cells were fixed and stained
with rhodamine-phalloidin. Images were obtained using a ?100
objective.
Densitometry of immunoblot. Densitometry of bands obtained by
immunoblot was performed using the software ImageJ developed by
the National Institutes of Health. Each band was normalized against
the corresponding ?-actin, GAPDH, or total Rac1 loading control.
These values were normalized to the control condition. For zymog-
raphy, the image was inverted, so that the white band became black,
and densitometry was assessed as described above.
Statistical analysis. Values are means ? SE. Measurements were
made on at least three independent wells from at least two different
isolations (ATII cells) or experiments (MLE12). A t-test was used
when only two groups were compared, and one-way ANOVA with
Bonferroni’s method was used for comparisons of multiple treatments
Fig. 2. ATII and MLE12 cells express CXCR4. Immunofluorescence shows expression of CXCR4 (green) in ATII (A and C) and MLE12 (D) cells, with nuclei
indicated by DRAQ5 staining (blue). C: cells migrating at a wound edge. B and E: Western blots of CXCR4 from 3 different isolations of ATII cells (B) and
3 different passages of MLE12 cells (E). Lysate from Jurkat 6.1 cells was used as a positive control, and ?-actin was used as loading control.
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to determine significant differences between individual conditions.
Significant differences were determined based on a threshold of P ?
0.05. Statistical comparisons were made using Sigmastat 3.5 (Jandel
Scientific, San Rafael, CA).
RESULTS
CXCL12 was secreted following injury. To determine whether
CXCL12 secretion was increased following injury, we mea-
sured CXCL12 in the BAL of rats ventilated with an injurious
tidal volume for 4 h. As shown in Fig. 1, CXCL12 in the BAL
was significantly increased in ventilated rats compared with
control (nonventilated) rats. We then measured the secretion of
CXCL12 from primary rat ATII cells and from MLE12 cells.
When cells were injured by multiple scratch wounds, the
concentration of CXCL12 was significantly higher in the con-
ditioned medium than in medium from unwounded control
cells.
ATII and MLE12 cells express the chemokine receptor
CXCR4. To demonstrate the expression of CXCR4 in ATII
cells and MLE12 cells, we used immunofluorescence (Fig. 2,
A, C, and D). These images show clear expression of CXCR4
in freshly isolated ATII cells (Fig. 2, A and C) and in confluent
cultures of MLE12 cells (Fig. 2D). Figure 2C shows CXCR4
expression at the leading edge of wounded ATII cells. To
further support these findings, Western blots from total cell
lysates demonstrate CXCR4 expression in ATII cells from
three different isolations (Fig. 2B) and in MLE12 cells from
different passages (Fig. 2E).
Blocking CXCR4 impeded wound healing in vitro. To deter-
mine whether CXCR4 has a functional role in alveolar epithe-
lial repair mechanisms, we measured cell migration in response
to CXCL12 (100 ng/ml) in a scratch-wound model. CXCL12
significantly increased wound closure by 16 h in ATII cells
(Figs. 3, A and B). Next, we examined the effect of treatment
with a specific blocker of CXCR4, AMD-3100, on wound
closure. AMD-3100 inhibited wound closure of ATII (Fig. 3, C
and D) and MLE12 (Fig. 3, E and F) cells in a dose-dependent
manner. AMD-3100 at ?0.5 ?g/ml caused significant inhibi-
tion in both cell types. To confirm that the inhibition was not
due to cell toxicity, we examined cell viability by Trypan blue
exclusion. We found that cell viability was 93.8 and 94.2% in
control ATII and MLE12 cells, respectively, while cell viabil-
ity following 24 h of treatment with 5 ?g/ml AMD-3100 was
94.5 and 93.9% (Fig. 3, C and E).
Knockdown of CXCR4 with shRNA attenuated wound
healing. To further investigate the role of CXCR4 in cell
migration, we developed a stable MLE12 cell line expressing
shRNA against CXCR4 (CXCR4-shRNA cells). Figure 4
shows that expression of CXCR4 was significantly de-
creased in these cells by ?80% compared with cells ex-
pressing a scrambled shRNA. Figure 4A is a representative
immunoblot of CXCR4 from control and CXCR4-shRNA
cells, and Fig. 4B demonstrates the specificity of the CXCR4
immunoblot with a CXCR4 blocking peptide. The knock-
down shown in Fig. 4C remained consistent in consecutive
passages. Figure 4, D and E, shows that wound closure was
Fig. 3. CXCL12 stimulated wound healing,
and blocking of CXCR4 impeded wound
healing. Scratch wounds were made on con-
fluent monolayers of ATII (A–D) and MLE12
(E and F) cells, and cells were treated with
CXCL12 (A and B) or AMD-3100 (C–F).
Wound widths were measured after 16 or 24
h on 3 fields per well and averaged. A, C, and
E: images of initial and final wounds; B, D,
and F: data expressed as percentage of orig-
inal wound width at 16 or 24 h. CXCL12
(100 ng/ml) stimulated wound closure in
ATII cells.Treatment
caused a dose-dependent inhibition of wound
closure in ATII and MLE12 cells. Values are
means ? SE (n ? 6). *P ? 0.05 vs. untreated
control. Images of untreated cells and cells
treated with 5 ?g/ml AMD-3100 for 24 h and
stained with Trypan blue (C and E) indicate
no effect on cell viability.
with AMD-3100
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significantly inhibited in CXCR4-shRNA cells compared
with control cells.
CXCR4 regulates MMP-2, MMP-14, and tissue inhibitor of
metalloproteinase-3. MMPs are important regulators of the
ECM and are typically activated during epithelial repair pro-
cesses (59). CXCR4 activation has been previously linked to
the regulation of MMPs (49). To determine whether inhibition
of CXCR4 affected activity of MMPs, we performed zymog-
raphy experiments using ATII cells and shRNA cell lines.
Figure 5A shows that blocking of CXCR4 with AMD-3100
reduced the activity of an MMP at 66 kDa in the conditioned
medium from ATII cells. Using Western blotting, we screened
cell lysate and supernatant samples for MMP-1, MMP-2,
MMP-3, MMP-9, MMP-11, MMP-12, MMP-13, MMP-14,
and MMP-21. We found immunoreactivity to MMP-2 in the
conditioned media and cell lysates of ATII and MLE12 cells at
?60–72 kDa, which encompasses the active (?62–66 kDa)
and pro (?72 kDa) forms of MMP-2 (Fig. 5B). We also found
immunoreactivity to MMP-14 in the lysate of cells at ?60–72
kDa, which would include pro-MMP-14, but little immunore-
activity was found in the conditioned medium in the expected
range for pro-MMP-14 (?66 kDa) or active MMP-14 (?54 kDa).
MMP-14 is known to cleave pro-MMP-2 to active MMP-2. To
determine whether decreased expression of MMP-14 would cause
reduced MMP-2 activity, we used siRNA against MMP-14 in rat
ATII cells. As shown in Fig. 6, A and B, expression of MMP-14
was decreased by ?60%, and this led to a significant decrease in
MMP-2 activity in the conditioned medium (Fig. 6, A and C).
Furthermore, decreased expression of MMP-14 caused a signifi-
cant inhibition of wound closure (Fig. 6D).
Fig. 4. Knockdown of CXCR4 with short hairpin RNA (shRNA) attenuated wound healing. CXCR4 was knocked down in MLE12 cells using shRNA.
A and C: decreased CXCR4 expression in CXCR4-shRNA compared with control shRNA MLE12 cell line and densitometry summarized from 5 sets of
blots. GAPDH was used as a loading control. B: specificity of immunoblot for CXCR4 with addition of a CXCR4 blocking peptide. D: initial and
final images of wounds in control shRNA and CXCR4-shRNA cells. E: wound closure at 24 h. Values are means ? SE (n ? 6). *P ? 0.05 vs. control
shRNA.
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Previous reports suggest that MMP-2 and MMP-14 activa-
tion can be regulated by tissue inhibitor of metalloproteinase-3
(TIMP-3) (5, 22, 25). Therefore, we assayed the expression of
TIMP-3 in control and AMD-3100-treated ATII cells. Inhibi-
tion of CXCR4 caused a significant increase in TIMP-3 ex-
pression (Fig. 7A). When we examined the basal activity of
MMP-2 in control MLE12 and CXCR4-shRNA cell lines, we
found a significant reduction in MMP-2 activity in the CXCR4-
shRNA cells (Fig. 7B). Similarly, TIMP-3 expression was
significantly increased in CXCR4-shRNA cells compared with
control cells (Fig. 7C).
CXCL12 activates the small GTPase Rac1. Rac1 was pre-
viously reported to regulate actin polymerization and cell
migration in response to CXCL12 (23). After 2 min of treat-
ment with CXCL12, we found that Rac1 activity was signifi-
cantly increased in ATII cells, and AMD-3100 blocked this
increase (Fig. 8, A and B). Treatment with CXCL12 also
increased Rac1 activity in control shRNA cells, but this did not
occur in CXCR4-shRNA (Fig. 8, C and D). To determine
whether changes in Rac1 activity would affect MMP-2 activ-
ity, we treated MLE12 cells with the Rac1 inhibitor W56 and
performed zymography using the conditioned medium. MMP-2 ac-
tivity was significantly decreased in the cells treated with Rac1
inhibitor (Fig. 9B), but there was no change in total Rac1 levels
(Fig. 9C).
CXCR4 regulates actin structure at the wound edge. To
investigate the role of CXCR4 on actin structure in migrating
cells at the leading edge of a wound, we visualized actin using
Alexa Fluor 594-labeled phalloidin on a confocal microscope.
As we showed previously, a prominent band of actin forms at
the leading edge of migrating cells (Fig. 10, A and C). When
ATII cells were treated with AMD-3100, this band was lost
(Fig. 10B). This band was also less prominent in CXCR4-
shRNA cells (Fig. 10D).
Fig. 5. CXCR4 regulates matrix metalloproteinase (MMP)-2 and MMP-14 in
ATII and MLE12 cells. A: zymography from conditioned media collected from
untreated ATII cells and cells treated with 2 ?g/ml AMD-3100 for 24 h.
Densitometry from 3 experiments was normalized to untreated control.
B: conditioned media and cell lysates from ATII and MLE12 cells were run for
PAGE in 4–12% gradient Bis-Tris gels, and immunoblot was performed using
different antibodies of MMPs. Of the MMPs screened (MMP-1, MMP-2,
MMP-3, MMP-9, MMP-11, MMP-12, MMP-13, MMP-14, and MMP-21),
only MMP-2 and MMP-14 showed strong reactivity. GAPDH was used as a
loading control for cell lysates. Medium was conditioned by cells seeded at
equal densities and with an equivalent volume. Jurkat 6.1 cells were used as a
positive control for MMP-2 and MMP-14.
Fig. 6. Knockdown of MMP-14 caused de-
creased MMP-2 activity and inhibited wound
closure. MMP-14 was knocked down in rat
ATII cells using small interfering RNA
(siRNA). A and B: siRNA caused signifi-
cantly decreased expression of MMP-14 in 3
different isolations. A and C: MMP-2 activity
was significantly decreased by siRNA against
MMP-14. D: MMP-14 knockdown caused a
significant decrease in wound closure. Values
are means ? SE (n ? 3). A scrambled siRNA
sequence was used as a control for all studies.
*P ? 0.05 vs. control siRNA.
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DISCUSSION
ATII cells have stem cell-like characteristics that facili-
tate the repair of the alveolar epithelium following injury (1,
14, 36, 51). One of the most important events in the early
stages of epithelial repair is the migration of ATII cells to
cover the denuded area, which is followed by later prolif-
eration and differentiation. Bone marrow-derived hemato-
poietic stem cells also contribute to the repair of injured
lungs, and these cells are recruited to the site of injury by
chemokines such as CXCL12 (11, 27, 52, 53, 60). While the
role of chemokine signaling is well recognized in the re-
cruitment of stem cells and immune cells, the role of
CXCL12/CXCR4 signaling in the migration of ATII cells
has not been previously shown. We showed for the first time
that CXCL12 secretion was increased in wounded cells in
culture and in the BAL of rats ventilated with a high tidal
volume (Fig. 1). We then confirmed that primary cultures of
rat ATII cells and MLE12 cells express CXCR4 (Fig. 2). We
showed that CXCL12 stimulated ATII cell migration and
that inhibition or knockdown of CXCR4 caused decreased
migration of ATII and MLE12 cells, respectively (Figs. 3
and 4). Our shRNA data demonstrated that the knockdown
level of CXCR4 was ?80%, but wound healing was inhibited
by ?30%. Thus the magnitude of downmodulation of CXCR4
by shRNA did not translate functionally to loss of cell migra-
tion, suggesting that other CXCR4-independent pathways are
involved in wound repair. It is also possible that proliferation
accounted for a significant portion of wound closure, which
may not have been affected by CXCR4.
To investigate the mechanisms by which CXCR4 stimulated
migration, we used zymography to determine whether MMP
activity was altered by CXCR4 inhibition or knockdown.
Secretion and activation of MMPs are important components
of cell migration, as MMPs digest the ECM and promote a path
for migrating cells. Interestingly, our data revealed that inhi-
bition or knockdown of CXCR4 caused decreased expression
of MMP-14 and decreased activity of MMP-2 (Figs. 5 and 7).
MMP-2 and MMP-14 (also known as MT1-MMP) have im-
portant roles in lung repair and development, as activation of
MMP-2 by MMP-14 has been shown in lung cells, and knock-
out mice exhibit abnormal airway and alveolar structures (3,
29, 31, 41). MMP-14 function was recently linked to CXCR4
activity during metastasis of melanoma cells to the lung (6),
and independent and coordinated roles for these molecules
were identified. Our results suggest that loss of function of
CXCR4 in ATII cells decreased the secretion of active
MMP-2, but we did not determine whether inhibition or down-
modulation of MMP-2 would affect CXCR4. We did show that
knockdown of MMP-14 caused decreased MMP-2 activity and
inhibited wound closure (Fig. 6).
Fig. 7. CXCR4 regulates tissue inhibitor of metallo-
proteinase (TIMP)-3 expression and MMP-2 activity
in ATII and MLE12 cells. A: expression of TIMP-3
in ATII cells in the presence and absence of AMD-
3100 and densitometric analysis. ?-Actin was used
as loading control. B: zymography and densitometric
analysis from control shRNA and CXCR4-shRNA
MLE12 cells. C: TIMP-3 expression in control
shRNA and CXCR4-shRNA MLE12 cells. Values
are means ? SE (n ? 3). *P ? 0.05 vs. control.
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In conjunction with decreased CXCR4 activity, we also
observed an increase in TIMP-3 expression (Fig. 7). TIMP-3 is
a strong regulator of several MMPs, including MMP-14,
MMP-2, and MMP-9 (5, 22, 25). Unlike TIMP-1 and TIMP-2,
TIMP-3 remains adherent to the ECM and accelerates cellular
transformation and proliferation (61). TIMP-3 plays an impor-
tant role in the regulation of airway branching (26). Our data
demonstrated a significant increase of TIMP-3 when CXCR4
Fig. 8. CXCL12 activates the small GTPase
Rac1. ATII (A and B) and MLE12 (C and D)
cells were treated with or without CXCL12
(100 ng/ml) for 2 min in the presence or
absence of AMD-3100 (AMD, 2 ?g/ml for 2
h prior to CXCL12). Cell lysates were col-
lected, and Rac1 activity was assayed. Den-
sitometry was performed and values were
normalized by dividing the level of activated
(Act) Rac1 by the total Rac1 level and then
normalizing to GTP control level. Values are
means ? SE; n ? 3 (A) and 4 (B). *P ? 0.05
vs. control cells. #P ? 0.05 vs. CXCL12-
treated cells.
Fig. 9. Inhibition of Rac1 activity decreased
MMP-2 activity. MLE12 cells were treated with
or without W56 (50 ?M) for 24 h, and condi-
tioned media were collected for zymography (A
and B) and cell lysates were collected for mea-
surement of total Rac1 (C). Densitometric anal-
ysis shows that inhibition of Rac1 caused de-
creased activity of MMP-2, but total Rac1 levels
were unchanged. Values are means ? SE; n ?
3. *P ? 0.05 vs. control.
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was inhibited or downregulated, suggesting that CXCR4
caused loss of MMP-14 expression and MMP-2 activity
through increased TIMP-3 expression.
A limitation of our study is that we did not evaluate the
effect of CXCL12/CXCR4 on proliferation during wound
closure. We previously demonstrated that ?13% of the cells
near the wound edge of cultured ATII cells had incorporated
ethidium deoxyuridine (as an index of proliferation) by 24 h
compared with ?4% of unwounded cells (13). Thus we cannot
rule out the possibility that CXCL12/CXCR4 may also affect
cell proliferation. Since ATII cells spread, migrate, proliferate,
and differentiate into type I cells in vivo, our model is limited,
and the effects of CXCL12/CXCR4 on alveolar repair should
be assessed in vivo. Our model also does not incorporate the
complex architecture of the alveolus, in which ATII cells do
not exist as a flat monolayer of cells.
Epithelial cell migration is associated with dramatic actin
remodeling and cytoskeletal rearrangement. We previously
identified that RhoA and Rac1 are important regulators of cell
migration in bronchial and alveolar epithelial cells (15–17).
Since CXCR4 activation has been shown to cause induction of
Rac1 activity in lymphocytes (4, 23), we showed that Rac1 was
stimulated by CXCL12 and that this could be blocked by
AMD-3100 or CXCR4 knockdown (Fig. 8). As discussed
above, Bartolome et al. (6) showed that migration of melanoma
cells was dependent on functional interactions between CXCR4 and
MMP-14, and they also showed that this involved Rac1. We
found that inhibition of Rac1 caused decreased MMP-2 activity
without affecting the overall expression of Rac1 (Fig. 9).
CXCL12-mediated stimulation of intestinal epithelial cell mi-
gration was found to involve ERK1/2 MAP kinase and phos-
phatidylinositol 3-kinase signaling and increased F-actin accu-
mulation at the leading edge of wounded monolayers (38, 50).
We showed that inhibition or knockdown of CXCR4 inhibited
the formation of a thick actin band at the leading edge of
migrating cells (Fig. 10). Our results suggest that CXCR4
activation following wounding causes a Rac1-dependent in-
crease in MMP-2 activity. Mice deficient in Rac2 expression
exhibit decreased levels of MMP-2 and MMP-9 (21), and
treatment of mice with a Rac1/Rac2 inactivator (8-oxo-2=-
deoxyguanosine)causeddiminishedactivityofMMP-2and MMP-9
in lungs (32). Our results elucidate a novel role of the chemo-
kine receptor CXCR4 in ATII cell migration and show that
CXCR4 regulates TIMP-3 and MMP-14 expression, Rac1-
dependent MMP-2 activation, and actin remodeling.
ACKNOWLEDGMENTS
We are grateful to Charlean Luellen for technical assistance.
GRANTS
These studies were supported by National Heart, Lung, and Blood Institute
Grant HL-094366 (C. M. Waters).
DISCLOSURES
No conflicts of interest, financial or otherwise are declared by the authors.
AUTHOR CONTRIBUTIONS
M.C.G. and C.M.W. are responsible for conception and design of the
research; M.C.G., P.S.M., and V.K.G. performed the experiments; M.C.G. and
C.M.W. analyzed the data; M.C.G., P.S.M., V.K.G., S.E.S., and C.M.W.
interpreted the results of the experiments; M.C.G. and C.M.W. prepared the
figures; M.C.G. and C.M.W. drafted the manuscript; M.C.G., P.S.M., S.E.S.,
and C.M.W. edited and revised the manuscript; M.C.G. and C.M.W. approved
the final version of the manuscript.
Fig. 10. CXCR4 regulates actin structure at the wound
edge. ATII (A and B) and control shRNA MLE12 (C)
and CXCR4-shRNA (D) cells were stained with Alexa
Fluor 594-labeled phalloidin 12 h after wounding. A
and C: prominent actin bands (arrowheads) in un-
treated ATII cells and control shRNA MLE12 cells,
respectively. B: CXCR4 inhibition with AMD-3100 (2
?g/ml) caused reduced organization of the actin band
at the leading edge. D: a similar effect by reduced
expression of CXCR4 in CXCR4-shRNA cells.
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