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ORIGINAL ARTICLE
Extracellular calumenin suppresses ERK1/2 signaling and cell
migration by protecting fibulin-1 from MMP-13-mediated
proteolysis
Q Wang
1,3
, B Shen
1,3
, L Chen
1,3
, P Zheng
1,3
, H Feng
1
, Q Hao
1
, X Liu
1
, L Liu
1
,SXu
1
, J Chen
1,2
and J Teng
1
Extracellular proteins are vital for cell activities, such as cell migration. Calumenin is highly conserved among eukaryotes, but its
functions are largely unclear. Here, we identify extracellular calumenin as a suppressor of cell migration and tumor metastasis.
Calumenin binds to and stabilizes fibulin-1, leading to inactivation of extracellular signal-regulated kinases 1 and 2 (ERK1/2)
signaling. We further identify the minimal functional domain of calumenin (amino acids 74–138 and 214–280). Depletion of
calumenin induces fibulin-1- and phospho-ERK1/2 (pERK1/2)-dependent promotion of cell migration. Consistently, in hepatocellular
and pancreatic carcinoma, both calumenin and fibulin-1 are downregulated. Furthermore, we show that matrix metalloproteinase-
13 (MMP-13) proteolyzes fibulin-1 and that calumenin protects fibulin-1 from cleavage by MMP-13. Calumenin, together with
fibulin-1, also interacts with fibronectin and depends on both syndecan-4 and α5β1-integrin to suppress ERK1/2 signaling and
inhibit cell migration. Thus, extracellular calumenin regulates fibulin-1 to have crucial roles in ERK1/2 signaling and cell migration.
Oncogene advance online publication, 17 March 2014; doi:10.1038/onc.2014.52
INTRODUCTION
Cell migration is a fundamental process in embryonic develop-
ment and pathological conditions such as tumor metastasis and
inflammation. In the extracellular space, extracellular matrix (ECM)
proteins form a complicated structural and functional network,
which not only provides structural support for cells, but also
integrates complex, multivalent signals to regulate various
physiological processes, like cell migration.
1
Fibulin-1 is a secreted
glycoprotein, which interacts with many other ECM proteins, such
as fibronectin, laminin, fibrinogen, aggrecan and versican,
2–4
and
is thus considered as an adhesion bridge of ECM structures.
1
Fibulin-1 inhibits cell adhesion and migration via fibronectin and
the receptor syndecan-4,
4,5
suppresses angiogenesis possibly
through angiogenin
6,7
and regulates cardiac ventricular morpho-
genesis via versican and ErbB2,
8
suggesting that fibulin-1
regulates cell adhesion and migration. However, the molecular
mechanism underlying the functional regulation of fibulin-1 in
extracellular space is largely unknown.
Matrix metalloproteinases (MMPs) are the major extracellular
proteinases that regulate ECM homeostasis and remodeling.
9
MMP-13 is a collagenase capable of cleaving rigid collagen fibrils
to remodel ECM structures, as well as to release embedded
signaling molecules.
10
Besides, MMP-13 also cleaves other
substrates, such as gelatin, aggrecan, perlecan and tenacin-C.
11
The expression of MMP-13 is influenced by a wide range of
cytokines and hormones, is commonly upregulated in tumors and
is correlated with cancer metastasis and aggressiveness.
11–13
Furthermore, MMP-13 is secreted as an immature precursor, and
undergoes activation by auto-proteolysis or peptide removal by
several other MMPs.
14
However, how MMP-13 is regulated
extracellularly after maturing remains to be determined.
Human calumenin, a member of the CREC protein family,
15
is
encoded by the CALU gene (NCBI GeneID: 813).
16
Fifteen
alternative splice variants are identified as calumenin-1–15, with
calumenin-1/2 (shown as calumenin without specific indication)
being the most abundant.
17–19
Calumenin contains a signal
peptide and is detectable in the extracellular space.
20,21
Proteomic
analysis suggests that calumenin is downregulated in MHCC97-H
cells, a human hepatocellular carcinoma (HCC) cell line with high
metastatic potential, compared with low metastatic potential
MHCC97-L cells.
22
Similarly, a lower expression level of calumenin
was also reported in human head and neck squamous cell
carcinoma
23
and lung squamous cell carcinoma.
24
Nevertheless,
the physiological function of extracellular calumenin, and whether
there is a correlation between calumenin expression and tumor
malignancy remain unclear. Here we show that calumenin
protects fibulin-1 from MMP-13-mediated proteolysis, and thus
inhibiting extracellular signal-regulated kinases 1 and 2 (ERK1/2)
phosphorylation and cell migration.
RESULTS
Extracellular calumenin inhibits cell migration
We first tested the effect of calumenin on cell migration.
Overexpression of calumenin-1/2-enhanced green fluorescent
protein (EGFP) markedly inhibited wound healing in HeLa
cells (Supplementary Figure S1a). Mutation of asparagine to alanine
at position 131 (N131A) of calumenin-2, which was reported to be
1
State Key Laboratory of Bio-Membrane and Membrane Bio-Engineering, Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, Department of Cell
Biology and Genetics, College of Life Sciences, Peking University, Beijing, China and
2
Center for Quantitative Biology, Peking University, Beijing, China. Correspondence: Dr J Chen
or Dr J Teng, State Key Laboratory of Bio-Membrane and Membrane Bio-Engineering, Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, College
of Life Sciences, Peking University, 5 Yiheyuan Road, Haidian District, Beijing 100871, China.
E-mail: chenjg@pku.edu.cn or junlinteng@pku.edu.cn
3
These authors contributed equally to this work.
Received 31 October 2013; revised 6 January 2014; accepted 20 January 2014
Oncogene (2014), 1–13
© 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14
www.nature.com/onc
an N-glycosylation site
20
and important for its stability,
21
resulted in
failure to inhibit wound healing (Supplementary Figure S1a). To test
whether it is the extracellular calumenin that impaired cell
migration, we added conditioned medium from calumenin-1/2-
EGFP-expressing HEK293T cells to HeLa cells, and this also inhibited
wound healing of HeLa cells (Figures 1a and b), while had no
detectable influence on cell proliferation indicated by BrdU labeling
(Supplementary Figure S1b). Transwell assays confirmed that
extracellular calumenin-1/2-EGFP suppressed cell migration of
HeLa (Figure 1c) and MDA-MB-231 cells (Supplementary Figure
S1c). To exclude the potential steric hindrance of the C-terminal
EGFP tag, we used calumenin-1/2-internal ribosome entry site
(IRES)-EGFP in Matrigel-coated transwell assays, and found that the
numbers of HeLa cells migrated through the transwell were also
significantly decreased (Figure 1d). Furthermore, addition of
purified calumenin-1-His-Strep to the medium of HeLa cells further
substantiated that extracellular calumenin suppressed cell migra-
tion (Figure 1e).
Calumenin inhibits cell migration by fibulin-1
Q Wang et al
2
Oncogene (2014), 1 –13 © 2014 Macmillan Publishers Limited
To investigate the effect of calumenin on cell migration and
invasion in vivo, we used the classical B16 melanoma mouse
model.
25
We first generated B16/F10 melanoma cell lines
stably expressing calumenin-1/2-IRES-EGFP (Supplementary
Figure S1d). Wound-healing assays showed that calumenin-1/2
overexpression also suppressed B16/F10 cell migration
(Supplementary Figure S1e). We then injected these cells into
mice through the tail vein and observed their lungs 2 weeks
later. The number of pulmonary metastatic colonies formed by
calumenin-1/2-overexpressing cells was significantly fewer than
that by the control cells (Figure 1f and Supplementary Figure S1f),
while no significant difference in the xenograft tumor
weight (Supplementary Figure S1g) and volume (Supplementary
Figure S1h) were observed when subcutaneously transplanting
these cell lines into nude mice.
Then, loss-of-function of endogenous calumenin-1/2, either
by blockade with its antibody (Figure 1g) or by knockdown with
small interfering RNA (siRNA; Figures 1h and i) in HeLa cells,
markedly promoted cell migration, while addition of condi-
tioned medium containing calumenin-1/2 significantly rescued
the phenotype induced by knockdown of calumenin-1/2
(Figure 1h and i). Consistently, knockdown of calumenin-1/2 in
B16/F0 cells also promoted cell migration (Supplementary
Figures S2a–c), and increased the number of pulmonary
metastatic colonies (Figure 1j). Together, these findings reveal
that extracellular calumenin suppresses cell migration both
in vitro and in vivo.
Minimal functional domain of calumenin for inhibiting cell
migration
To determine the minimal functional domain of calumenin-1, we
first tested the effects of 15 isoforms of calumenin on cell
migration, and found that calumenin-1, 2, 3, 4, 5 and 7
inhibited HeLa cell migration (Supplementary Figure S3a). After
analyzing the exon composition of these isoforms, we further
generated 14 EGFP-tagged truncates, named calumenin-1-Δ1to
calumenin-1-Δ14 (Figure 2a), and transfected them into HeLa
cells. Wound-healing assays showed that these truncates fell
into two categories, one inhibiting cell migration like the
full-length calumenin-1-EGFP, while the other having no
significant effect (Figures 2b and d). The two shortest truncates,
calumenin-1-Δ13 (amino acids 74–138 and 214–315) and
calumenin-1-Δ14 (amino acids 74–138 and 214–280) were
confirmed to suppress cell migration by transwell assays
(Figure 2e). To validate the identified minimal functional domain
of calumenin-1 in vivo, we generated B16/F10 melanoma cell
lines stably expressing calumenin-1-Δ14-EGFP (Supplementary
Figure S3b), which showed reduced wound-healing efficiency
(Supplementary Figure S3c). We then injected them into mice
through the tail vein. The number of pulmonary metastatic
colonies formed by these cells was significantly fewer than that
of the control group (Figure 2f and Supplementary Figure S3d).
Thus, the minimal functional domain, calumenin-1-Δ14, is
sufficient to suppress cell migration.
Extracellular calumenin inhibits cell migration via suppressing
ERK1/2 phosphorylation
The mitogen-activated protein kinase signaling pathway is
known as a main signaling pathway to regulate cell
migration,
26
thus we tested the three major mitogen-activated
protein kinases: ERK1/2, p38 and JNK. Overexpression of
calumenin-1/2 suppressed the level of phospho-ERK1/2 (pERK1/2),
but not p-p38 or pJNK, in HeLa (Figure 3a) and B16/F10
melanoma cells (Figure 3b). Consistently, both siRNA knock-
down (Figure 3c) and antibody blockade of calumenin-1/2
(Figure 3d) promoted ERK1/2 phosphorylation, and the
pERK1/2 level in siRNA-treated cells was restored by adding
conditioned medium containing calumenin-1/2 (Figure 3c).
In addition, a decrease of pERK1/2 was detected 7 h
after addition of the eukaryotically expressed and purified
calumenin-1 to the medium of HeLa cells (Figure 3e).
Similar results were observed for pMEK1/2 (Figure 3f), the
upstream kinase of ERK1/2. We then asked whether the
inhibitory effect of calumenin on cell migration was
pERK1/2 dependent. In the presence of U0126, an inhibitor of
ERK1/2 phosphorylation,
27
blockade of calumenin by its
antibody failed to promote cell migration (Figure 3d). Moreover,
the minimal functional domain of calumenin, calumenin-1-Δ13
and calumenin-1-Δ14, also decreased the pERK1/2 level
(Figure 3g). Together, these data suggest that the inhibition of
cell migration by extracellular calumenin is dependent on
ERK1/2 activation.
ERK1/2 regulates actin polymerization and stimulates the
formation of lamellipodia to promote cell migration.
28,29
To
examine the effect of calumenin on cell morphology, we labeled
actin filaments along with paxillin, a marker of focal adhesion.
30
Figure 1. Extracellular calumenin suppresses cell migration. (a) Images of wound-healing assays of HeLa cells at 0, 24 and 48 h post-
scratching, in the presence of conditioned medium containing HEK293T-cell-derived SP19-EGFP or calumenin-1/2-EGFP. EGFP-tagged
protein in conditioned medium of HEK293T cells was detected by western blot using anti-EGFP antibody at 48 h post-transfection.
(b)Quantification of wound sizes from (a)(n=8). (c) Transwell assays of HeLa cells, with HEK293T cells in the lower chamber transfected
with SP19-EGFP or calumenin-1/2-EGFP. Numbers of HeLa cells migrated to the lower side of the transwells are shown (n=4). EGFP-tagged
protein in conditioned medium from HEK293T cells were detected by western blot using anti-EGFP antibody at 48 h post-transfection.
(d) Invasion assays of HeLa cells with HEK293T cells in the lower chamber expressing IRES-EGFP or calumenin-1/2-IRES-EGFP. Numbers of
HeLa cells migrated to the lower side of the transwells are shown (n=4). Calumenin-1/2 in conditioned medium from HEK293T cells were
detected by western blot at 48 h post-transfection. (e) Transwell assays of HeLa cells treated with eukaryotically expressed and purified
calumenin-1-His-Strep (1 μg/ml). Numbers of HeLa cells migrated to the lower side of the transwells are shown (n=4). (f) B16/F10 cells
stably expressing IRES-EGFP or calumenin-1/2-IRES-EGFP were injected into C57BL/6J mice via the tail veins. Representative lungs and
quantification of the numbers of metastatic pulmonary colonies 2 weeks post-injection are shown (n=12). (g) Transwell assays of HeLa cells
treated with normal IgG or anti-calumenin-1/2 antibody (5 μg/ml). Numbers of HeLa cells migrated to the lower side of the transwells are
shown (n=4). (h) RNA interference efficiency of HeLa cells transfected with control or calumenin-1/2 siRNA for 72 h. HEK293T cells derived
calumenin-1/2-containing conditioned medium was concentrated and added for rescue experiments. Calumenin-1/2 in total cell lysates
and conditioned medium were detected by western blot. (i) Transwell assays of HeLa cells corresponding to (h). Numbers of HeLa cells
migrated to the lower side of the transwells are shown (n=4). (j) B16/F0 cells stably expressing control or calumenin-1/2 short hairpin RNA
(shRNA) were injected into C57BL/6J mice via the tail veins. Representative lungs and quantification of the numbers of metastatic
pulmonary colonies 3 weeks post-injection are shown (n=15 or 14). For a,c,d,e,g,i, scale bars, 100 μm; for f,j, scale bars, 1 cm. 293T,
HEK293T cells; Calu, calumenin; CM, conditioned medium; Co.St., Coomassie blue staining; SP19, signal peptide (amino acids 1–19) of
calumenin; TCL, total cell lysates. For b–e,g,i,dataaremean±s.d.; for f,j,dataaremean±s.e.m., *Po0.05, **Po0.01, ***Po0.001,
determined by unpaired two-tailed Student’st-test.
Calumenin inhibits cell migration by fibulin-1
Q Wang et al
3
© 2014 Macmillan Publishers Limited Oncogene (2014), 1 –13
In both HeLa (Figure 3h) and B16/F10 cells (Figures 3h and i),
the elevation of calumenin-1/2 protein level induced a
smooth cell periphery and promoted the cells to aggregate in
a cobblestone-like formation. Conversely, calumenin-1/2 knock-
down in B16/F0 cells resulted in an elongated fibroblast-like
morphology with pseudopodia-like protrusions (Figures 3j and k).
Thus, calumenin induces cell migration-related morphology
changes.
Calumenin interacts with fibulin-1
To screen for calumenin-interacting proteins involved in the
ERK1/2 pathway, in the extracellular space or on the plasma
membrane, we performed immunoprecipitation assays of
total cell lysates and conditioned medium of HEK293T cells
expressing calumenin-1-EGFP. The bound proteins were
separated by sodium dodecyl sulfate–polyacrylamide gel
electrophoresis and analyzed by liquid chromatography with
Figure 2. The minimal functional truncate of calumenin. (a) Schematic pictures of the architecture of 14 calumenin-1 truncates. aa,
amino-acid; +/, truncates suppressing/not suppressing cell migration in wound-healing assays. (b) Expression levels of 14 calumenin-1-
EGFP truncates in HeLa cells were detected by western blot using anti-EGFP antibody or reverse-transcript PCR (RT–PCR) 24 h
post-transfection. (c) Wound-healing assays of HeLa cells expressing the EGFP-tagged calumenin-1 truncates at 0, 12, 24, 48 and 72 h
post-scratching. Scale bar, 100 μm. (d)Quantification of wound sizes from (c)(n=8). (e) Transwell assays of HeLa cells, with HEK293T cells
in the lower chamber transfected with calumenin-1-EGFP or calumenin-1-Δ13/14-EGFP. Numbers of HeLa cells migrated to the lower side
of the transwells are shown (n=4). EGFP-tagged protein in conditioned medium from HEK293T cells was detected by
western blot using anti-EGFP antibody at 48 h post-transfection. (f) B16/F10 cells stably expressing SP-EGFP or calumenin-1-Δ14-EGFP
were injected into C57BL/6J mice via the tail veins. Representative lungs and quantification of the numbers of metastatic pulmonary
colonies 2 weeks post-infection are shown (n=13). Scale bar, 1 cm. Calu, calumenin; SP19, signal peptide (amino acids 1–19)
of calumenin. For d,e, data are mean ±s.d.; for f,dataaremean±s.e.m., *Po0.05, ***Po0.001, determined by unpaired two-tailed
Student’st-test.
Calumenin inhibits cell migration by fibulin-1
Q Wang et al
4
Oncogene (2014), 1 –13 © 2014 Macmillan Publishers Limited
tandem mass spectrometry. Many potential binding proteins
were identified (Supplementary Figures S4a–c), among which
fibulin-1 was the most abundant (Figure 4a). As it was also
reported to inhibit ERK1/2 phosphorylation,
4
we focused on
fibulin-1.
Fibulin-1 has four isoforms produced by alternative mRNA
splicing (Supplementary Figure S4b) with fibulin-1D being the
longest.
31
We then used fibulin-1D (described as fibulin-1, unless
otherwise indicated) and confirmed its interaction with calum-
enin-1/2 by immunoprecipitation in conditioned medium (Figures
4b and c). Furthermore, both in vitro binding assays (Figure 4d)
and yeast two-hybrid assays (Supplementary Figure S4d) showed
that calumenin-1/2 directly interacted with fibulin-1. In addition,
the minimal functional domain of calumenin, calumenin-1-Δ13
and calumenin-1-Δ14, also interacted with fibulin-1 (Figure 4e),
suggesting that calumenin-1-Δ14 (amino acids 78–134 and
218–280) are sufficient for its interaction with fibulin-1. However,
the diminished interaction of calumenin minimal functional
truncates and fibulin-1 suggested that the other parts of
calumenin may also facilitate its interaction with fibulin-1
(Figure 4e). We then generated EGFP-tagged truncation mutants
of fibulin-1 to map its interaction domain with calumenin.
Calumenin-2-glutathione S-transferase co-immunoprecipitated
with the full-length fibulin-1 and its truncates fibulin-1Δ4,
Δ5 and Δ6 (Figure 4f). All these truncates contained EGF-like
repeats 7–9, suggesting these repeats (amino acids 441–578) of
fibulin-1 are crucial for its interaction with calumenin. Collectively,
calumenin directly interacts with fibulin-1 in the extracellular
space, and amino acids 78–134 and 218–280 of calumenin and
EGF-like repeats 7–9offibulin-1 are required for their interaction.
Calumenin stabilizes fibulin-1 and both calumenin and fibulin-1
are inversely correlated with hepatocellular and pancreatic
carcinoma
Next, we tested the relationship between calumenin and fibulin-1
in cell migration. Blockade of fibulin-1 by its antibody significantly
reduced the suppressive activity of calumenin-1 on HeLa cell
migration (Figure 5a), and effectively blocked the calumenin-1-
induced reduction of ERK1/2 phosphorylation (Figure 5b). More-
over, after knockdown of fibulin-1, no significant difference was
detected between control and calumenin-1-overexpressing B16/F10
cells in wound-healing assays (Supplementary Figures S4e and f).
Consistently, the number of pulmonary metastatic colonies
formed by these cells did not show any significant difference
(Figure 5c). All these data suggest that calumenin suppresses cell
migration in a fibulin-1-dependent manner.
We noticed that the suppressive ability on cell migration was
more efficient in cells co-expressing fibulin-1 and calumenin-1
than the expression of each alone (Supplementary Figure S4g),
implying that calumenin and fibulin-1 function synergistically to
inhibit cell migration. Interestingly, we found that depletion of
calumenin-1/2 either by siRNA (Figure 5d and Supplementary
Figure S2a) or antibody (Figure 5e) reduced the fibulin-1 protein
level, whereas overexpression of calumenin-1/2 markedly
enhanced it in the extracellular space (Figures 3b and 5f), with
its mRNA level unchanged (Supplementary Figure S4h). The
minimal functional domain of calumenin, calumenin-1-Δ13 and
calumenin-1-Δ14, also enhanced the fibulin-1 protein level
(Figure 5g). Together, these data suggest that calumenin stabilizes
fibulin-1 in the extracellular space.
Proteins that inhibit cell migration are frequently downregu-
lated in cancer.
32
Considering that both calumenin and fibulin-1
inhibit cell migration, we sought to determine whether they were
simultaneously downregulated in cancer cells and tumor tissues
from patients. Both the calumenin-1/2 and fibulin-1 protein levels
were downregulated, while the pERK1/2 level was elevated in
high metastatic potential MHCC97-H cells, compared with low
metastatic potential MHCC97-L cells (Supplementary Figure S5a).
Consistently, immunohistochemical staining showed decreased
calumenin-1/2 and fibulin-1 in most HCC tissues (Figure 5h). In
detail, in samples from 75 HCC patients (Supplementary Figure
S5b and Supplementary Table S1), ~ 65% showed a decrease of
both calumenin-1/2 and fibulin-1 in tumor tissues (Figure 5i).
Moreover, patients with higher HCC grades (II–III) had lower
protein levels of both calumenin-1/2 and fibulin-1 than those with
lower grades (I–II) (Supplementary Figures S5c-e). Similar to HCC,
both calumenin-1/2 and fibulin-1 displayed clearly lower expres-
sion in pancreatic tumor tissues compared with their adjacent
normal tissues (Figure 5j and Supplementary Table S2). In samples
from 74 patients with pancreatic carcinoma, ~ 54% showed a
decrease of both proteins (Figure 5k and Supplementary Figure
S5f). Together, these results reveal that both calumenin and
fibulin-1 are significantly downregulated in HCC and pancreatic
carcinoma.
Calumenin protects fibulin-1 from cleavage by MMP-13
ECM proteins are usually proteolyzed by MMPs,
9
leading us to
hypothesize that calumenin protects fibulin-1 from MMP-
mediated proteolysis. However, no MMP has been identified to
cleave fibulin-1. Therefore, we first overexpressed several MMPs
(MMP-9, -13, -14, -15 and -19) in HeLa cells, and found that only
MMP-13 overexpression decreased the extracellular fibulin-1
protein level (Figure 6a). Consistently, in vitro cleavage assays
using the purified proteins also showed that MMP-13–3×flag
cleaved fibulin-1-HA, leading to the appearance of specific cleaved
bands (Figure 6b). Therefore, these data suggest that fibulin-1 is a
substrate of MMP-13.
To investigate the effect of calumenin on MMP-13-mediated
fibulin-1 degradation, we co-overexpressed calumenin-1 and
MMP-13, and found that calumenin-1 suppressed the cleavage
of fibulin-1 protein by MMP-13 (Figure 6c). Meanwhile, neither
MMP-13 nor calumenin-1 influenced the mRNA level of fibulin-1
(Supplementary Figure S4h). Further in vitro cleavage assays also
demonstrated that the cleavage of fibulin-1 by MMP-13 was
suppressed in the presence of calumenin-1-EGFP (Figure 6d).
To further confirm the relationships of calumenin, fibulin-1 and
MMP-13 on cell migration, we performed transwell assays.
MMP-13 enhanced cell migration, while blockade of fibulin-1 by
its antibody eliminated this enhancement, suggesting that
MMP-13 functions in a fibulin-1-dependent manner (Figure 6e).
Importantly, overexpression of calumenin-1-EGFP abolished the
MMP-13-mediated promotion of cell migration (Figure 6f).
Together, these data reveal that MMP-13 degrades fibulin-1 to
facilitate cell migration, while calumenin binds to fibulin-1 and
protects it from MMP-13-induced cleavage to inhibit cell
migration.
Calumenin inhibits cell migration through fibronectin, syndecan-4
and α5β1-integrin
Fibulin-1 was reported to inhibit cell migration via its association
with fibronectin,
4
leading us to investigate the relationships
among fibronectin, fibulin-1 and calumenin. Overexpressed
calumenin-1-EGFP interacted with both endogenous fibulin-1
and fibronectin (Figure 4b), and endogenous calumenin-1/2 and
fibulin-1 were co-immunoprecipitated with fibronectin (Figure 7a),
suggesting that calumenin forms a complex with fibulin-1 and
fibronectin. Besides, calumenin-1/2-EGFP overexpression led to
more fibulin-1 co-immunoprecipitated with fibronectin
(Figure 7b). Blockade of calumenin-1/2 by its antibody facilitated
cell migration, however, in the presence of anti-fibronectin
antibody, anti-calumenin-1/2 antibody failed to enhance cell
migration (Figure 7c), suggesting that fibronectin is indispensable
for calumenin and fibulin-1 to inhibit cell migration.
Calumenin inhibits cell migration by fibulin-1
Q Wang et al
5
© 2014 Macmillan Publishers Limited Oncogene (2014), 1 –13
Previous work showed that fibulin-1 inhibits fibroblast cell
adhesion through competing with syndecan-4 to bind to
fibronectin, thus inhibiting cell adhesion and migration.
5
There-
fore, we examined whether syndecan-4 is required for the
function of calumenin to inhibit cell migration. The enhanced cell
migration induced by anti-calumenin-1/2 antibody treatment was
abolished by adding anti-syndecan-4 antibody to the medium
(Figure 7d). Moreover, the effect of antibody against calumenin on
Calumenin inhibits cell migration by fibulin-1
Q Wang et al
6
Oncogene (2014), 1 –13 © 2014 Macmillan Publishers Limited
cell migration was also antagonized by antibodies against α5β1-
integrin (Figure 7e), a major cell surface receptor of fibronectin
33
that synergistically function with syndecans in cell migration and
adhesion.
34,35
Although more studies are needed to address their
relationships, these data suggest that the inhibition of cell
migration by calumenin depends on fibronectin, syndecan-4 and
α5β1-integrin.
In conclusion, calumenin directly interacts with fibulin-1,
protects it from cleavage by MMP-13, and enhances the binding
of fibulin-1 to fibronectin. Through syndecan-4 and α5β1-integrin
receptors, the formation of a calumenin/fibulin-1/fibronectin
complex leads to inhibition of the ERK1/2 signaling pathway
and the subsequent suppression of cell migration (Figure 7f).
DISCUSSION
ECM remodeling is a key event of cell migration, and the activity of
extracellular proteases, especially MMPs, has crucial roles in this
process.
9
Among MMPs, MMP-13 is highly expressed in tumor and
stroma tissues, and is a suitable target of anti-cancer and anti-
inflammation drugs.
13,36,37
Previous studies showed that MMP-13
activity is mainly regulated at three distinct levels: transcription,
Figure 3. Calumenin suppresses ERK1/2 phosphorylation and regulates actin organization. (a,b) Western blot of total cell lysates and
conditioned medium from HeLa (a) or B16/F10 (b) cells overexpressing the indicated proteins for 48 h. Quantification of relative pERK1/2 to
ERK1/2 levels (a) are shown (n=3). (c) Western blot of total cell lysates and conditioned medium from HeLa cells at 72 h post-transfection with
control or calumenin-1/2 siRNA. HEK293T cells derived calumenin-1/2-containing conditioned medium was concentrated and added for
rescue experiments. (d) Transwell assays of HeLa cells treated with MEK inhibitor U0126 (10 μM) and anti-calumenin-1/2 antibody (5 μg/ml).
Numbers of HeLa cells migrated to the lower side of the transwells are shown (n=4). Protein levels of total cell lysates from the treated HeLa
cells were analyzed by western blot using anti-pERK1/2 and anti-calumenin antibodies. (e,f) pERK1/2 (e) and pMEK1/2 (f) of HeLa cells treated
with eukaryotically expressed and purified calumenin-1-His-Strep (1 μg/ml) were detected by western blot. (g) pERK1/2 of HeLa cells
overexpressing calumenin-1-EGFP or calumenin-1-Δ13/14-EGFP at 24 h post-transfection were detected by western blot. (h) Co-staining of
paxillin and actin filaments in HeLa cells overexpressing SP19-EGFP or calumenin-1/2-EGFP for 72 h. (i,j) Co-staining of paxillin and actin
filaments (i) or differential interference contrast images (j) of B10/F10 cells stably expressing IRES-EGFP or calumenin-1-IRES-EGFP.
(k,l) Co-staining of paxillin and actin filaments (k) or differential interference contrast images (l) of B10/F0 cells stably expressing control short
hairpin RNA (shRNA) or calumenin-1/2 shRNA. For h,i,k, scale bars, 10 μm; for j,l, scale bars, 100 μm. Calu, calumenin; CM, conditioned
medium; Co.St., Coomassie blue staining; SP19, signal peptide (amino acids 1–19) of calumenin-1/2; TCL, total cell lysates. For a,d, data are
mean ±s.d., **Po0.01, ***Po0.001, determined by unpaired two-tailed Student’st-test.
Figure 4. Calumenin interacts with fibulin-1 in the extracellular space. (a) Immunoprecipitation assays with anti-EGFP antibody in conditioned
medium and total cell lysates from HEK293T cells expressing calumenin-1-EGFP. Immunoprecipitates were subjected to sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and Coomassie blue staining. Arrows indicate the proteins identified by liquid
chromatography with tandem mass spectrometry (LC-MS/MS) analysis. (b) Conditioned medium from HEK293T cells expressing SP19-EGFP or
calumenin-1/2-EGFP was immunoprecipitated with anti-EGFP antibody, and the immunoprecipitates were analyzed with anti-fibulin-1, anti-
fibronectin and anti-EGFP antibodies by western blot. (c) Conditioned medium from HeLa cells was immunoprecipitated with anti-fibulin-1
antibody, and the immunoprecipitates were analyzed with anti-calumenin-1/2 and anti-fibulin-1 antibodies by Western blot, using horseradish
peroxidase (HRP)-conjugated protein A as the secondary antibody. (d)In vitro binding assay of calumenin-1-EGFP (purified with anti-EGFP
antibody) and fibulin-1-HA (purified with anti-HA antibody). The purified proteins were mixed and immunoprecipitated with anti-EGFP
antibody. The immunoprecipitates were then analyzed by western blot with anti-HA antibody. (e) Conditioned medium from HeLa cells
overexpressing calumenin-1-Δ13/14-EGFP was immunoprecipitated with anti-EGFP antibody, and the immunoprecipitates were analyzed with
anti-fibulin-1 antibody by western blot. (f) Mapping the interacting domains of calumenin-2-glutathione S-transferase (GST ) and EGFP-tagged
fibulin-1 truncates. HEK293T cells overexpressing full-length calumenin-2-GST and EGFP-tagged fibulin-1 truncates were immunoprecipitated
with anti-EGFP antibody, and analyzed with anti-GST antibody by western blot. Asterisks indicate bands of fibulin-1 truncates. Schematic
pictures of fibulin-1 truncates are shown in the left panel. +/ , binding positive/negative. 293T, HEK293T cells; Calu, calumenin; CM,
conditioned medium; Co.St., Coomassie blue staining; IP, immunoprecipitation; SP19, signal peptide (amino acids 1–19) of calumenin; TCL,
total cell lysates.
Calumenin inhibits cell migration by fibulin-1
Q Wang et al
7
© 2014 Macmillan Publishers Limited Oncogene (2014), 1 –13
post-translational activation and interaction with inhibitors.
11,38
However, as far as we know, no protein has been reported to bind
to MMP-13 substrates to inhibit MMP-13-mediated cleavage. Here,
we show that fibulin-1 cleavage by MMP-13 is blocked by
calumenin. Whether calumenin directly inhibits MMP-13 activity,
or protects fibulin-1 from MMP-13 access remains to be
investigated. Nevertheless, our findings open a new dimension
in the understanding of the regulation of MMP-13. We propose
that combined treatment with calumenin and inhibitors of
MMP-13 may serve as a new therapeutic strategy for tumor
metastasis. Besides, calumenin interacts with fibronectin, which is
also a substrate of MMP-13.
39
It is possible that calumenin also
inhibits MMP-13-mediated proteolysis of fibronectin to inhibit cell
migration, which needs further investigation.
Fibulin-1 assists in the formation of macromolecular ECM
structures,
1
and also inhibits cell migration.
4
Here, we first
identify fibulin-1 as a substrate of MMP-13, and detect MMP-13-
digested bands of fibulin-1 (Figure 6b). We propose that multiple
Figure 5. Calumenin stabilizes fibulin-1, and both calumenin and fibulin-1 protein levels are inversely correlated with HCC and pancreatic
carcinoma. (a) Transwell assays of HeLa cells in the presence of calumenin-1-IRES-EGFP from HEK293T cells, and simultaneously treated with
normal IgG or anti-fibulin-1 antibody. Numbers of HeLa cells migrated to the lower side of the transwells are shown (n=4). Data are mean ±s.d.,
**Po0.01, ***Po0.001, determined by unpaired two-tailed Student’st-test. (b) HeLa cells were transfected with SP19-EGFP or calumenin-1-
EGFP, and simultaneously treated with normal IgG or anti-fibulin-1 antibody (5 μg/ml) for 24 h. Total cell lysates were analyzed with anti-
pERK1/2 antibody by western blot. (c) B16/F10 cells stably co-expressing fibulin-1 shRNA and calumenin-1 were injected into C57BL/6J mice
via the tail veins. Representative lungs and quantification of the numbers of metastatic pulmonary colonies 2 weeks post-injection are shown
(n=15 or 13). Data are mean±s.e.m., n.s., not significant, determined by unpaired two-tailed Student’st-test. (d,e) HeLa cells were transfected
with control or calumenin-1/2 siRNA for 72 h (d), or treated with normal IgG or anti-calumenin-1/2 antibody (5 μg/ml) for 24 h (e). Total cell
lysates and conditioned medium were analyzed with anti-calumenin and anti-fibulin-1 antibodies by western blot. (f,g) HeLa cells were
transfected with calumenin-1/2-IRES-EGFP (f) or calumenin-1-Δ13/14-EGFP (g) for 48 h. Fibulin-1 in conditioned medium were analyzed by
western blot. (h,j) Immunohistochemical staining of calumenin-1/2 and fibulin-1 in HCC tissues (h), pancreatic carcinoma tissues (j) and their
corresponding adjacent normal tissues. Scale bars, 100 μm. (i,k) Statistical analysis of the immunohistochemical staining signal intensity of
calumenin-1/2 and fibulin-1 in tumor samples from 75 HCC patients (i) and 74 pancreatic tumor patients (k). ↓/↑, expression level decreased/
increased in tumor tissue; , no clear change detected; green box, simultaneous decrease of both calumenin-1/2 and fibulin-1. 293T, HEK293T
cells; Calu, calumenin; CM, conditioned medium; Co.St., Coomassie blue staining; SP19, signal peptide (amino acids 1–19) of calumenin; TCL,
total cell lysates.
Calumenin inhibits cell migration by fibulin-1
Q Wang et al
8
Oncogene (2014), 1 –13 © 2014 Macmillan Publishers Limited
MMP-13 cleavage sites exist on fibulin-1, which is consistent
with the previous reports that MMPs tend to cleave substrates
at many sites.
40
As these bands (mostly >50 kDa) are recognized
by anti-HA antibody, and the HA epitope is tagged at the
C-terminus of fibulin-1, we speculate that some of the MMP-13
cleavage sites are located in its N-terminus. Considering that
calumenin is associated with EGF-like repeats 7–9 on the
C-terminus of fibulin-1, it is unlikely that calumenin simply shields
the MMP-13 cleavage sites on fibulin-1, but rather stabilizes its
whole structure.
Figure 6. Calumenin inhibits MMP-13-mediated fibulin-1 proteolysis. (a) Fibulin-1 were detected by western blot in conditioned medium of
HeLa cells overexpressing different MMPs. Expression levels of MMPs were detected by RT–PCR. (b)In vitro cleavage assays of fibulin-1-HA
(purified by anti-HA antibody) by MMP-13–3×flag (purified by anti-flag antibody, and activated by its activator APMA). 1 × , 2× , 4 × , 8× and
16 × indicate increasing concentration of MMP-13. Cleaved bands of fibulin-1 are marked by asterisks. (c) HeLa cells were co-expressed with
calumenin-1-EGFP and MMP-13–3×flag for 48 h. Total cell lysates and conditioned medium were analyzed with anti-fibulin-1, anti-EGFP, and
anti-flag antibodies by western blot. (d)In vitro cleavage assays of fibulin-1-HA (purified by anti-HA antibody) by MMP-13–3×flag (purified by
anti-flag antibody, and activated by its activator APMA), in the presence or absence of calumenin-1-EGFP (purified by anti-EGFP antibody). The
cleavage samples were analyzed with anti-HA antibody by western blot. (e) Transwell assays of HeLa cells overexpressing MMP-13–3×flag,
and simultaneously treated with normal IgG or anti-fibulin-1 antibody (5 μg/ml). Numbers of HeLa cells migrated to the lower side of
transwells are shown (n=4). MMP-13–3×flag in conditioned medium from HEK293T cells were detected by western blot using anti-flag
antibody. (f) Transwell assays of HeLa cells co-expressing calumenin-1-EGFP and MMP-13–3×flag. Numbers of HeLa cells migrated to the
lower side of transwells are shown (n=4). EGFP- or flag-tagged protein in conditioned medium from HeLa cells were detected by western
blot using anti-EGFP or anti-flag antibody, respectively. APMA, 4-aminophenylmercuric acetate; Calu, calumenin; CM, conditioned medium;
Co.St., Coomassie blue staining; SP19, signal peptide (amino acids 1–19) of calumenin; TCL, total cell lysates. For e,f, data are mean ±s.d.,
**Po0.01, ***Po0.001, n.s. not significant, determined by unpaired two-tailed Student’st-test.
Calumenin inhibits cell migration by fibulin-1
Q Wang et al
9
© 2014 Macmillan Publishers Limited Oncogene (2014), 1 –13
Our results, as well as previous reports, show that the full-length
fibulin-1 inhibits cell migration,
5
while no such inhibition occurs
after its degradation by MMP-13. However, the MMP-digested
fragments of some ECM proteins bind to and activate cell surface
receptors.
33
Therefore, whether the MMP-13-digested fragments
of fibulin-1 have regulatory roles in cell migration or in other
physiological process is worth further investigation.
In HCC and pancreatic carcinoma tissues, we find that
calumenin and fibulin-1 are inversely correlated with tumor grade
and metastatic potential. A similar situation of calumenin is also
described in human head and neck squamous cell carcinoma
23
and lung squamous cell carcinoma.
24
A recent report showed that
fibulin-1 mRNA and protein levels are reduced in human HCC
samples.
41
These data further strengthen that calumenin and
fibulin-1 suppress tumor metastasis. However, progressive expres-
sion of calumenin is reported in colon
42
and endometrial
carcinomas.
43
Similarly, elevated fibulin-1 are also detected in
ovarian and breast carcinomas,
44,45
and fibulin-1C may participate
in ovarian tumorigenesis,
46
suggesting that the inverse association
between calumenin, fibulin-1 and tumor metastasis may be
tissue specific.
MATERIALS AND METHODS
Cell lines and tumor samples
HeLa, HEK293T, MCF-7, MDA-MB-231, MHCC97-L and MHCC97-H cells were
cultured in Dulbecco’s modified Eagle’s medium (GIBCO, Grand Island, NY,
USA), B16/F10 and B16/F0 in RPMI 1640 (GIBCO) and supplemented with
10% fetal bovine serum (GIBCO) at 37 °C with 5% CO
2
.
Reagents and antibodies
Reagents used were: 4-aminophenylmercuric acetate (Merck, Whitehouse
Station, NJ, USA), BrdU (Calbiochem, Darmstadt, Germany), ERK1/2
inhibitor U0126 (Promega, Madison, WI, USA), G418 (GIBCO), horseradish
Figure 7. Calumenin inhibits cell migration through fibronectin, syndecan-4 and α5β1-integrin. (a) Conditioned medium from HeLa cells was
immunoprecipitated with anti-fibronectin antibody, and the immunoprecipitates were analyzed with anti-fibulin-1 and anti-calumenin
antibodies by western blot. (b) Conditioned medium from HEK293T cells overexpressing SP19-EGFP or calumenin-1/2-EGFP was
immunoprecipitated with anti-fibronectin antibody. The immunoprecipitates were analyzed by western blot with anti-fibulin-1 antibody.
(c–e) Transwell assays of HeLa cells in the presence of anti-calu-1/2 antibody (5 μg/ml), together with anti-fibronectin antibody (5 μg/ml) (c), or
anti-syndecan-4 antibody (5 μg/ml) (d), or anti-β1 and anti-α5-integrin antibodies (5 μg/ml) (e). Numbers of HeLa cells migrated to the lower
side of transwells are shown (n=4). (f) A schematic model illustrating that calumenin suppresses MMP-13-mediated proteolysis of fibulin-1 in
the extracellular space to regulate cell migration. Calumenin-1/2 protects fibulin-1 from MMP-13-mediated proteolysis, and the stabilized
fibulin-1 binds to fibronectin, which possibly decreases the binding of integrins and syndecans-4 to fibronectin, and thus inhibits the
syndecan-integrin-ERK1/2 signaling pathway and cell migration. Calu, calumenin; CM, conditioned medium; Co.St., Coomassie blue staining;
IP, immunoprecipitation; SP19, signal peptide (amino acids 1–19) of calumenin; TCL, total cell lysates. For c–e, data are mean ±s.d.,
***Po0.001, n.s. not significant, determined by unpaired two-tailed Student’st-test.
Calumenin inhibits cell migration by fibulin-1
Q Wang et al
10
Oncogene (2014), 1 –13 © 2014 Macmillan Publishers Limited
peroxidase-conjugated protein A (Sigma-Aldrich, St Louis, MO, USA), JetPEI
(Polyplus, Illkirch, France), Phalloidin (Atto 647N; Sigma-Aldrich), puromycin
(Sigma-Aldrich), Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and
Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). Rabbit anti-EGFP
polyclonal antibody were self-produced. Antibodies of α5-integrin (Abcam,
Cambridge, UK, ab75472), β1-integrin (Abcam, ab24693), calumenin
(ProteinTech, Wuhan, China, 17804–1-AP), BrdU (MBL, MI-11–3, Woburn,
MA, USA), EGFP (MBL, M0483), ERK1/2 (Santa Cruz, Dallas, TX, USA, sc-154),
phospho-ERK1/2 (Santa Cruz, sc-7383), fibronectin (Abcam, ab23750),
fibronectin (BD Biosciences, 610078), fibulin-1 (Abcam, ab54652), fibulin-1
(3A11, was provided by W Scott Argraves),
4
fibulin-1 (Santa Cruz, sc-20818),
flag (Sigma-Aldrich, F1804), HA (Sigma-Aldrich, H9658), JNK (Cell Signaling,
Danvers, MA, USA, 9252), phospho-JNK (Cell Signaling, 9251), MEK1/2
(Epitomics, Burlingame, CA, USA, S2436), phospho-MEK1/2 (Epitomics,
1470–1), p38 (Cell Signaling, 9212), phospho-p38 (Cell Signaling, 9216),
paxillin (BD Biosciences, 610052) and sydecan-4 (Santa Cruz, sc-12766)
were used. The secondary antibodies Alexa Fluor 488-/568-conjugated
goat anti-mouse/rabbit IgG (Invitrogen) and horseradish peroxidase-
conjugated antibodies (Jackson ImmunoResearch, West Grove, PA, USA)
were used.
Vector construction
Human calumenin-1/2 and fibulin-1D transcripts were amplified from HeLa
cell complementary DNA. MMP-9, -13, -14, -15 and -19 were amplified from
HeLa or HEK293T cell complementary DNA. Short hairpin RNAs were
constructed into pLKO.1 vector (Sigma-Aldrich).
Wound-healing and two-chamber transwell assays
Wound-healing assays were performed as previously reported.
47
For
transwell assays, 6.5-mm Millicell inserts (Millipore, Billerica, MA, USA) were
used as previously reported.
48
HEK293T or MCF-7 cells in the lower
chamber were transfected with plasmids, and 24 h post-transfection, HeLa
or MDA-MB-231 cells (2 × 10
5
cells) were added into the upper chamber of
each Millicell. For antibody blockade, antibodies (5 μg/ml) were added into
the lower chamber. After 6–8 h, cells that migrated to the lower surface
were stained with haematoxylin and eosin. Samples were examined under
an IX71 inverted microscope (Olympus, Tokyo, Japan) and statistically
calculated.
RNA interference and lentivirus package
For calumenin knockdown, HeLa cells were transfected with an siRNA pool
against calumenin (see Supplementary Table S3 for target sequences).
Total cell lysates and conditioned medium were collected and subjected to
western blot 72 h after transfection. For rescue assays, calumenin
expressed by HEK293T cells was added into conditioned medium 48 h
post-transfection. For lentivirus production, the pLKO.1 vector containing
short hairpin RNA was transfected into HEK293T cells together with
packaging plasmids, and virus was collected 72 h post-transfection and
used to infect target cells as previously reported.
49
Stable cell line generation
G418 (1 mg/ml) or puromycin (2 μg/ml) was added to B16/F10 or B16/F0
cells transfected with plasmids to kill the negative cells. Single cell with
green fluorescence was sorted into 96-well plates by flow cytometry
(Beckman Coulter, Brea, CA, USA) and cultured in the presence of G418 or
puromycin for 1–2 weeks. The proliferated cells were then subjected to
quantitative real-time–PCR and western blot analysis. At least two clones
were established for each cell line.
Animal studies
All animal experiments were undertaken in accordance with the National
Institute of Health Guide for the Care and Use of Laboratory Animals, and
with the approval of the Committee of Animal Research in Peking
University. Animals were kept in a specific pathogen-free environment in
the Peking University Laboratory Animal Center. For pulmonary coloniza-
tion mouse model, experiments were performed as previously described.
50
B16/F10 or B16/F0 stable cell lines (2–5×10
5
) were slowly injected into the
tail veins of female C57BL/6J mice (7 weeks old). After 2 or 3 weeks, the
in vivo imaging system FX Pro (Kodak, Rochester, NY, USA) was used to
image the EGFP fluorescence in living mice whose abdominal hair had
been shaved. The fluorescence intensity of the lung region was calculated.
Numbers of melanoma colonies in the lung were counted. For primary
tumor growth mouse model, B16/F10 cells (5 × 10
6
) suspended in Matrigel
were inoculated subcutaneously in the dorsal skinfold of BALB/c nude
mice (6 weeks old). Tumor length and width were measured on days 3, 6,
8, 9, 10, 11, 12 and 13 using calipers, and tumor volume was calculated as
π/6 × length × width
2
. On day 13 after inoculation, the mice were killed and
tumors were surgically removed and weighed.
Protein purification and in vitro experiments
For purification of calumenin-1–6 × His-Strep, HEK293T cells were trans-
fected with corresponding plasmids and maintained for 24 h followed by
replacement with serum-free Dulbecco’s modified Eagle’s medium for 48 h.
Total cell lysates and conditioned medium were harvested and purified by
sequential binding with Ni-NTA affinity chromatography (Invitrogen) and
Strep-Tactin columns (IBA, Göttingen, Germany).
For purification of calumenin-EGFP, fibulin-1-HA and proMMP-13–
3×flag, HEK293T cells transfected with corresponding plasmids were
maintained for 24 h followed by replacement with serum-free Dulbecco’s
modified Eagle’s medium for 48 h. Conditioned medium was harvested
and concentrated using Centricon YM-30 Concentrators (Millipore), and
used for immunoprecipitation. After washing with high-salt buffer (25 mM
HEPES, 2 mMCaCl
2
,1mMMgCl
2
, 500 mMNaCl, pH 7.4), the immunopre-
cipitated protein was eluted with HA or flag peptide, and used for in vitro
assays.
proMMP-13–3×flag was activated by treatment with 1 mM4-
aminophenylmercuric acetate for 45 min at 37 °C. The in vitro cleavage
was carried out in cleavage buffer (50 mMTris–HCl, 10 mMCaCl
2
, 150 mM
NaCl, 0.05% Brij-35, pH 7.5) at 37 °C for 24 h.
Immunofluorescence and histochemistry
Cells were fixed and labeled as previously described.
51
Paxillin was
immunostained with Alexa Fluor 568-conjugated secondary antibody, and
actin filaments were stained with Atto-647N-labeled phalloidin. Samples
were observed under an IX71 inverted fluorescence microscope (Olympus)
and a TCS SP2 confocal microscope (Leica, Solms, Germany) equipped with
a × 100/1.4 numerical aperture oil immersion objective lens.
Immunohistochemical staining of samples from HCC and pancreatic
carcinoma patients were performed by the National Engineering Center for
Biochip (Shanghai, China).
Immunoprecipitation, liquid chromatography with tandem mass
spectrometry and western blot
For identifying proteins interacting with calumenin, total cell lysates and
conditioned medium of HeLa cells transfected with calumenin-1-EGFP
were collected 48 h post-transfection. The samples were incubated with
anti-EGFP antibody overnight followed by conjugation with protein A
sepharose (Amersham, Pittsburgh, PA, USA) in immunoprecipitation buffer
(20 mMHEPES, 120 mMNaCl, 2 mMCaCl
2
,1mMMgCl
2
,1mMdithiothreitol,
0.1% Triton X-100, pH 7.4) with proteinase inhibitors. Samples were then
separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis.
Specific bands were excised and digested with sequence-grade modified
trypsin (Promega) and subjected to LCQ Deca XP Plus Analyzer liquid
chromatography/mass spectrometry (Finnigan, San Diego, CA, USA)
analysis.
Immunoprecipitation assays were performed as previously described.
52
For western blot assays, the proteins were transferred onto polyvinylidene
difluoride membranes (Millipore). Membranes were blocked and then
probed with primary and secondary antibodies.
RNA isolation and quantitative real-time–PCR
Total RNA was extracted with Tranzol reagent (Transgen, Beijing, China)
and reverse transcribed (Invitrogen) as in previous reports.
53,54
Quantita-
tive real-time–PCR was performed by an ABI 7300 detection system
(Applied Biosystems, Foster City, CA, USA) using SYBR Green PCR Master
Mix (Invitrogen) as previously described.
53
Glyceraldehyde 3-phosphate
dehydrogenase served as a reference control and the 2
ΔΔCT
method
55
was used (see Supplementary Table S3 for primers).
BrdU incorporation assay
Cells were incubated in 15 μMBrdU (Calbiochem) for 1 h before fixation,
permeabilized in 1% Triton X-100 and then treated with 1 Mand 2 MHCl
Calumenin inhibits cell migration by fibulin-1
Q Wang et al
11
© 2014 Macmillan Publishers Limited Oncogene (2014), 1 –13
sequentially. Cells were then neutralized and incubated with anti-BrdU
antibody. Finally, cells were labeled with 4,6-diamidino-2-phenylindole and
visualized under an IX71 inverted fluorescence microscope (Olympus).
Yeast two-hybrid assay
Yeast two-hybrid assays were performed using Matchmaker GAL4 two-
hybrid system 3 (Clontech, Mountain View, CA ,USA). pGBKT7-calumenin-2
and pGADT7-fibulin-1 were co-transformed into yeast strain AH109. The
yeasts were plated on SC-Trp-Leu plates and positive colonies were
subsequently plated on SC-Trp-Leu-His-Ade plates.
Statistical analysis
Student’st-test was used to analyze data.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
We thank Dr W Scott Argraves at the Medical University of South Carolina for kindly
providing anti-fibulin-1 antibody (3A11). The lentivirus system is a kind gift from
Dr Jincai Luo at Peking University. We also thank Dr Li Yu and Dr Xiaofeng Wang at
Tsinghua University, and Dr Zhengfan Jiang at Peking University for helpful
discussion. We also thank Dr IC Bruce at Zhejiang University and Dr Xiaolei Su at
the University of California, San Francisco, for reading the manuscript. This work was
supported by the National Natural Science Foundation of China (31271424) and the
Major State Basic Research Development Program of China (973 program)
(2010CB833705).
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