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OPEN
Calumenin-15 facilitates filopodia formation by
promoting TGF-bsuperfamily cytokine GDF-15
transcription
H Feng
1,3
, L Chen
1,3
, Q Wang
1,3
, B Shen
1
, L Liu
1
, P Zheng
1
,SXu
1
, X Liu
1
, J Chen
1,2
and J Teng*
,1
Filopodia, which are actin-rich finger-like membrane protrusions, have an important role in cell migration and tumor metastasis.
Here we identify 13 novel calumenin (Calu) isoforms (Calu 3–15) produced by alternative splicing, and find that Calu-15 promotes
filopodia formation and cell migration. Calu-15 shuttles between the nucleus and cytoplasm through interacting with importin a,
Ran GTPase, and Crm1. The phosphorylation of the threonine at position 73 (Thr-73) by casein kinase 2 (CK2) is essential for the
nuclear import of Calu-15, and either Thr-73 mutation or inhibition of CK2 interrupts its nuclear localization. In the nucleus, Calu-15
increases the transcription of growth differentiation factor-15 (GDF-15), a member of the transforming growth factor-b(TGF-b)
superfamily, via binding to its promoter region. Furthermore, Calu-15 induces filopodia formation mediated by GDF-15. Together,
we identify that Calu-15, a novel isoform of Calu with phosphorylation-dependent nuclear localization, has a critical role in
promoting filopodia formation and cell migration by upregulating the GDF-15 transcription.
Cell Death and Disease (2013) 4, e870; doi:10.1038/cddis.2013.403; published online 17 October 2013
Subject Category: Cancer
Filopodia, which are finger-like projections supported by
tightly parallel-bundled filamentous actin, are involved in
many key physiological and pathological processes,
1–3
whereas abundant filopodia are considered strongly correlated
with the enhanced cell migration and tumor metastasis.
3–5
Filopodia contain receptors for various extracellular signals,
including cytokines and growth factors, to sense the cell’s
surroundings during cell migration, then trigger the formation
of initial adhesion sites, the recruitment of focal adhesion
components, and finally the reorganization of the actin
network.
3,6–8
Transforming growth factor-b(TGF-b),
a cytokine, has been reported to induce the expression of
paxillin, a focal adhesion complex component, and fascin, an
actin filament-bundling protein, which in turn promotes
filopodia formation and cell migration.
4,7
Therefore, TGF-b
and fascin emerge as potential targets for cancer therapies.
4,9
Growth differentiation factor-15 (GDF-15) is a member of
the TGF-bsuperfamily proteins.
10,11
It is also known as
macrophage inhibitory cytokine-1, prostate-derived factor,
placental bone morphogenetic protein, non-steroidal
anti-inflammatory drug-activated gene-1, and placental
TGF-b.
10,11
Similar to other TGF-bsuperfamily members,
GDF-15 is first synthesized as a pro-protein, and then cleaved
and secreted in its active mature form.
12
It has been reported
to have roles in kinds of cellular processes such as cell
proliferation, migration, differentiation, and apoptosis.
13–17
GDF-15 has both anti- and pro-tumorigenic functions
according to different cell types and different developmental
stages in the tumor.
13,17,18
A high level of GDF-15 in serum is
associated with a poor patient survival in colorectal and
prostate carcinoma,
19,20
and the level of GDF-15 has also
been reported to increase during the transition of colonic
lesions to cancer initiation.
19
However, there is also evidence
that overexpression of GDF-15 induces the apoptosis of
breast cancer cells and inhibits the tumorigenicity of LN-Z308
glioblastoma cell line.
21,22
Therefore, to unravel the molecular
mechanisms that regulate GDF-15 is the key point for the
understanding and treatment of malignant tumor.
Calumenin (Calu) belongs to the CREC protein family,
which is composed of Cab45, reticulocalbin-1, reticulocalbin-2
(also known as ERC-55), reticulocalbin-3, and Calu.
23,24
These proteins all contain multiple EF-hand domains and are
encoded respectively by five genes.
23,24
Interestingly, most of
these genes produce isoforms by alternative mRNA splicing
and these isoforms usually have different subcellular localiza-
tions and physiological functions.
23,24
The CALU gene has
been reported to produce two isoforms, Calu-1 and
Calu-2 (also known as crocalbin),
25,26
having equal length
1
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, Beijing, China and
2
Center for Quantitative Biology, Peking University, Beijing, China
*Corresponding author: 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. Tel: þ86-10-62767044;
Fax: þ86-10-62755786; E-mail: junlinteng@pku.edu.cn
3
These authors contributed equally to this work.
Received 18.7.13; revised 04.9.13; accepted 11.9.13; Edited by G Melino
Keywords: calumenin-15; isoform; phosphorylation; GDF-15; transcription; filopodia
Abbreviations: Calu, calumenin; ChIP, chromatin immunoprecipitation; CK2, casein kinase 2; EGR-1, early growth response protein 1; FAK, focal adhesion kinase;
GDF-15, growth differentiation factor-15; LMB, leptomycin B; NLS, nuclear localization signal; NES, nuclear export signal; TBB, 4,5,6,7-tetrabromo-2-azabenzimidazole;
TGF-b, transforming growth factor-b
Citation: Cell Death and Disease (2013) 4, e870; doi:10.1038/cddis.2013.403
&
2013 Macmillan Publishers Limited All rights reserved 2041-4889/13
www.nature.com/cddis
(315 amino acids (aa)) with only exons 3 and 4 exchanged.
23
Recently, standard Edman degradation assay revealed that
both Calu-1 and Calu-2 possess an N-terminal signal peptide
(19 aa),
27
which leads to their translocations into the ER or
Golgi lumen.
28
Functionally, Calu-1 and Calu-2 regulate
vitamin K-dependent g-carboxylation activity
29
and participate
in calcium cycling by interacting with ryanodine receptor-1 and
ER Ca
2þ
-ATPase SERCA2.
30,31
Here we set out to determine whether there are other Calu
isoforms and identify that the CALU gene produces 13 novel
isoforms (named Calu 3–15) by alternative splicing, with only
one of them, Calu-15, possessing nuclear localization. Calu-
15 shuttles between the nucleus and cytoplasm, and this
process is regulated by its phosphorylation. Functionally,
Calu-15 increases GDF-15 transcription level in the nucleus,
which in turn induces filopodia formation and promotes cell
migration.
Results
Identification of Calu isoforms and their subcellular
localizations. To search for more alternatively spliced
isoforms of the CALU gene, we designed a pair of primers
localized in the first and last exon (Supplementary Figure
S1a), and performed PCR analysis. Several bands were
amplified from the cDNA of HeLa cells (Supplementary
Figure S1b), and 13 more splicing variants were identified by
sequencing. We named these 13 novel variants Calu 3–15
(GenBank accession number: HM002604–HM002616;
Figure 1a).
Previous reports have shown that Calu-1 and Calu-2 have
eight exons with mRNA lengths of B3.4 kb.
25,26
When the
exon organization of these isoforms was compared, we found
that Calu-3 and Calu-4 possessed a novel exon (exon 2 in
Figure 1a), leading to an increase of mRNA length to B4.2 kb.
In addition, Calu-3 and Calu-4 had extra 8 aa at the
N-terminus compared with Calu-1 and Calu-2
(Supplementary Figure S1c). As the N-terminal 19 aa of
Calu-1 and Calu-2 are the signal peptide,
27,28
we examined
whether the extra 8 aa interrupted this signal. The fluores-
cence assay showed that Calu-3–EGFP and Calu-4–EGFP
also colocalized with GRIP1-mRFP, a Golgi apparatus
marker, similar to Calu-1–EGFP and Calu-2–EGFP
(Figure 1b). Furthermore, EGFP fusion with the N-terminal
27 aa (8 þ19 aa) of Calu-3 and Calu-4 also colocalized with
GRIP1-mRFP (Supplementary Figure S1d). These results
suggest that the N-terminal 27 aa of Calu-3 and Calu-4 still
function as a signal peptide.
We also determined the subcellular distributions of the
other identified isoforms by overexpressing their EGFP fusion
proteins in HeLa cells. We found that only Calu-15 lacked the
signal peptide and showed nuclear accumulation (Figure 1b).
Together, the CALU gene encodes 15 isoforms, and only
Calu-15 lacks the N-terminal signal peptide and shows
nuclear localization.
Calu-15 shuttles between the nucleus and cytoplasm,
mediated by importin-a, Ran GTPase, and Crm1. To
confirm that Calu-15 shows the nuclear localization, we
tagged it with 2 EGFP and found that Calu-15–2 EGFP
was also localized in the nucleus (Figure 2a). Consistently,
b-Gal–EGFP-tagged Calu-15 also localized in the nucleus,
whereas the control b-Gal–EGFP did not (Supplementary
Figure S2). Thus, Calu-15 is able to drag 2 EGFP and
b-Gal–EGFP into the nucleus.
To determine the subcellular localization of endogenous
Calu-15, we used the antibody against Calu-1 and Calu-2,
which could also recognize the overexpressed Calu-15–
EGFP similar to the EGFP antibody (Figure 2b). Using this
antibody, we analyzed the nuclear and cytoplasmic fraction
proteins extracted from HeLa cells by western blotting, and
found that Calu-1 and Calu-2 were in the tubulin-containing
cytoplasmic fraction, whereas Calu-15 was mainly detected in
the nuclear fraction along with the nuclear marker lamin
(Figure 2c).
After confirming that Calu-15 is localized in the nucleus, we
next investigated whether it was through the classical path-
way to enter the nucleus. We performed immunoprecipitation
experiments and found that Calu-15 was associated with
importin-a(Figure 2d), a protein participating in the nuclear
entry process.
32,33
Besides, immunoprecipitation showed that
Calu-15 also interacted with Ran GTPase (Figure 2d), which
has been reported to disassociate the importin–cargo com-
plex and be required for the nuclear import process.
33,34
Moreover, the cotransfection with RanQ69L, which is a
mutant defective in GTPase activity,
35
largely inhibited the
nuclear localization of Calu-15 (Figure 2e). Therefore, the
nuclear import process of Calu-15 is mediated by importin-a
and regulated by Ran GTPase.
Interestingly, Calu-15 also interacted with the exportin
protein Crm1 (Figure 2d), which is reported to mediate
nucleocytoplasmic transport.
33,36
Therefore, it is reasonable
to hypothesize that Calu-15 might be exported from the
nucleus through a Crm1-dependent pathway. To test this, we
used leptomycin B (LMB), a small molecule that interferes
Crm1–cargo binding,
37
to suppress the Crm1-dependent
nuclear export. In cells overexpressing Calu-15–2 EGFP
due to its high level of nuclear accumulation, LMB treatment
could not increase the nuclear accumulation of Calu-15
(Figure 2e). However, in the presence of RanQ69L, LMB
treatment obviously induced Calu-15 to be localized in the
nucleus (Figure 2e). Thus, Calu-15 can shuttle between the
nucleus and cytoplasm, and this is mediated by importin-a,
Ran GTPase, and Crm1.
The nuclear localization of Calu-15 depends on phos-
phorylation at Thr-73 by CK2. Given that phosphorylation
can regulate the nuclear-cytoplasmic shuttling of proteins,
38,39
we analyzed the amino acid sequence of Calu-15
with bioinformatics tools
40
and found that the threonine at
position 73 (Thr-73) might be phosphorylated by casein
kinase 2 (CK2). This finding prompted us to examine whether
this site had a role in the nuclear-cytoplasmic shuttling of
Calu-15. We mutated this amino acid to alanine (T73A) and
glutamic acid (T73E) to mimic the unphosphorylated and
phosphorylated state, respectively, and examined their
subcellular localizations. To quantify the localization pattern,
we classified it into three types: (1) cells with predominantly
nuclear localization of EGFP fusion protein (N4C);
(2) with both nuclear and cytoplasmic localization (N¼C);
Calumenin-15 facilitates filopodia formation
H Feng et al
2
Cell Death and Disease
and (3) with predominantly cytoplasmic localization
(NoC). Compared with the wild-type Calu-15 (52.2% of N4C),
Calu-15-T73A showed no nuclear localization (0% of N4C),
whereas no significant difference was observed between
Calu-15-T73E and wild type (Figure 3a, P¼1.41 10
33
and
P¼0.10, respectively). Thus, these results suggest that the
phosphorylation of Calu-15 at Thr-73 is related to its nuclear
localization.
To confirm that Calu-15 is phosphorylated at Thr-73, we
used SDS-PAGE with 50 mM Phos-tag acrylamide, in which
phosphorylated bands migrate slower.
41
An upshifted phos-
phorylated band of Calu-15 was detected in wild type,
whereas this band almost disappeared in Calu-15-T73A
mutant (Figure 3b). Next, to determine whether it is CK2 that
phosphorylates the Thr-73 of Calu-15 and affects its nuclear
localization, we used 4,5,6,7-tetrabromo-2-azabenzimidazole
(TBB) to specifically inhibit CK2 activity
42
immediately after
the transfection of Calu-15–2 EGFP. TBB treatment
decreased the phosphorylation level of Calu-15 (Figure 3b),
and the nuclear localization of Calu-15 was also significantly
interrupted (from 75.3% to 32.3% of N4C) after 10 h TBB
treatment (Figure 3c, P¼5.27 10
10
). However, TBB
treatment did not change the subcellular localization of
Calu-15-T73E mutant (Figure 3c, P¼0.78), suggesting that
Figure 1 Fifteen calumenin (Calu) isoforms and their subcellular localizations. (a) Schematic picture shows the exon organization of 15 Calu isoforms encoded by the
CALU gene. The number at the top of the picture is the exon number. Black regions represent coding sequence, white regions represent untranslated mRNA sequence.
(b) Subcellular localizations of the enhanced green fluorescent protein (EGFP) fusion proteins of Calu isoforms (green). GRIP1-mRFP (red) marks the Golgi apparatus.
Bar, 10mm
Calumenin-15 facilitates filopodia formation
H Feng et al
3
Cell Death and Disease
CK2 promotes the nuclear localization of Calu-15 through
phosphorylation at Thr-73. As the loss of phosphorylation at
Thr-73 led to no nuclear localization of Calu-15 (Figure 3a), we
then examined whether it was due to the decrease of the
nuclear import or an increase of the nuclear export process.
The treatment of the nuclear export inhibitor LMB could not
increase the nuclear localization of Calu-15-T73A (Figure 3d,
P¼0.84), suggesting that the reason why the mutant
Calu-15–T73A showed no nuclear localization is not
the increase of nuclear export. Together, we conclude that
the phosphorylation of Calu-15 at Thr-73 by CK2 is
indispensable for its nuclear localization.
Calu-15 promotes GDF-15 transcription. To unravel the
physiological function of Calu-15 in the nucleus, we
examined the transcription levels of a group of genes
(Figure 4a). Quantitative real-time PCR analysis showed
that GDF-15 mRNA level was significantly increased in the
HeLa cell line stably expressing Calu-15–EGFP (Figure 4a,
P¼6.85 10
6
, and Supplementary Figure S3). Corre-
spondingly, GDF-15 protein level was also increased when
overexpressing Calu-15–2 EGFP, which is revealed by
western blotting (Figure 4b, P¼0.0006).
To investigate whether Calu-15 changes the GDF-15
expression level through interacting with its promoter, we
performed dual-luciferase reporter assay and found that
under the control of the promoter of GDF-15 (1 kb) the relative
luciferase activity was largely increased in Calu-15–EGFP-
expressing cells (Figure 4c, P¼3.67 10
–3
). Further analysis
showed that only a 200-bp region of the GDF-15 promoter was
sufficient (Figure 4c, P¼1.13 10
–3
). Moreover, ChIP assay
confirmed the interaction between Calu-15 and the promoter
Figure 2 Calumenin (Calu)-15 shuttles between the nucleus and the cytoplasm, mediated by importin-a, Ran GTPase, and Crm1. (a) Subcellular localization of Calu-15–
2enhanced green fluorescent protein (EGFP) fusion protein (green). Nuclear DNA was stained with DAPI (4’,6-diamidino-2-phenylindole; blue). Bar, 10 mm. The upper
panel shows the domain architecture of Calu-15–2 EGFP. (b) Immunoprecipitation (IP) with antibody against EGFP were performed in the lysates of HEK293T cells
expressing Calu-15–EGFP, followed by immunoblotting (western blotting (WB)) with antibody against Calu-1/2 (calu). Antibody against EGFP (EGFP) as a positive control.
IgG as a negative control. (c) Immunoblottings (WB) of endogenous Calu-15 in the cytoplasmic and nuclear fraction from HeLa cells. Asterisks indicate the band of Calu-1/2 or
Calu-15. Tubulin and lamin, respectively, labels the cytoplasmic and nuclear fraction. (d) IP with antibody against EGFP was performed in the lysates of HEK293T cells
expressing Calu-15–EGFP or EGFP, followed by immunoblotting (WB) with the indicated antibodies. (e) HeLa cells cotransfected with Calu-15–2 EGFP and RanQ69L-
mRFP were treated with (right panels) or without (left panels) leptomycin B (LMB) for 3 h, then fixed for observation. Nuclear DNA was stained with DAPI (blue). Bar, 10 mm
Calumenin-15 facilitates filopodia formation
H Feng et al
4
Cell Death and Disease
Figure 3 Casein kinase 2 (CK2) phosphorylates calumenin (Calu)-15 at threonine at position 73 (Thr-73) and facilitates its nuclear localization. (a) The subcellular
localization of Calu-15 wild-type (WT), T73A, or T73E. Left panels, representative pictures. Nuclear DNA was stained with DAPI (4’,6-diamidino-2-phenylindole; blue). Bar,
10 mm. Right panel, the graph shows the percentage of cells with predominantly nuclear (N4C), both nuclear and cytoplasmic (N ¼C) or predominantly cytoplasmic (NoC)
localization of enhanced green fluorescent protein (EGFP) fusion protein (n¼3; 450 cells per experiment). Data are mean±S.E.M.; ***Po0.001; n.s., no significant
difference (contingency table test for independence). (b) Lysates of HEK293T cells expressing Calu-15–2 EGFP WT or T73A mutants (upper panel), and lysates of
HEK293T cells expressing Calu-15–2 EGFP treated with dimethylsulfoxide (DMSO) or 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB; lower panel) were separated by Phos-
tag SDS-polyacrylamide gel electrophoresis, then immunoblotted with antibody against EGFP. Arrows indicate the phosphorylated form. (c) HeLa cells transfected with Calu-
15–2 EGFP WT or T73E mutant were treated with DMSO or TBB for 10 h post transfection and then fixed for observation. Left panels, representative pictures. Nuclear DNA
was stained with DAPI (blue). Bar, 10 mm. Right panel, the graphs show the percentage of cells with different localization patterns (n¼3; 450 cells per experiment). Data are
mean±S.E.M.; ***Po0.001; n.s., no significant difference (contingency table test for independence). (d) HeLa cells transfected with Calu-15–2 EGFP WT, T73A, or T73E
were treated with or without leptomycin B (LMB) before fixation and observation. Nuclear DNA was stained with DAPI (blue). Bar, 10 mm. The graphs show the percentage of
cells with different localization patterns (n¼3; 450 cells per experiment). Data are mean±S.E.M.; n.s., no significant difference (contingency table test for independence)
Calumenin-15 facilitates filopodia formation
H Feng et al
5
Cell Death and Disease
of GDF-15 (Figure 4d). To further verify our findings, we
showed that the non-phosphorylation mimic mutant T73A,
with cytoplasmic localization pattern, was insufficient to
increase the relative luciferase activity, which was under the
control of the 200-bp region of the GDF-15 promoter, whereas
both the wild-type and the phosphorylation mimic mutant
T73E could (Figure 4e, P¼0.0337). Together, these results
suggest that nuclear Calu-15 regulates the GDF-15 transcription
via binding to its promoter region.
Calu-15 facilitates filopodia formation through a GDF-15-
mediated pathway. Interestingly, we found that overexpres-
sion of Calu-15–EGFP promoted cell migration in the wound-
healing assay (Figure 5a and Supplementary Figure S4).
Besides, Calu-15 also significantly increased the number and
size of filopodia (Figure 5b, P¼9.59 10
–5
), which facilitates
cell migration through highly active actin rearrangement.
13
Accordingly, the phosphorylation mimic mutant T73E also
induced filopodia formation, whereas the unphosphorylation
mimic mutant T73A failed (Figure 5c, P¼1.46 10
–5
,
P¼5.03 10
6
and P¼3.84 10
5
, respectively). All
these observations are consistent with previous reports that
GDF-15 is involved in the regulation of cell migration.
13
Considering that GDF-15 is a cytokine and is secreted into
the extracellular space, we then tested the conditioned
medium from Calu-15–2 EGFP-expressing HEK293T cells
and the filopodia formation was also induced (Figure 5d,
P¼1.38 10
–4
). Moreover, we knocked down GDF-15 in
Calu-15–2 EGFP-overexpressing HeLa cells and found
that the depletion of GDF-15 inhibited the function of
Calu-15 on filopodia formation (Figure 5f, P¼9.20 10
–4
).
Collectively, these data suggest that Calu-15 can induce
filopodia formation through GDF-15, which in turn promotes
cell migration.
To further confirm the correlation among Calu-15,
GDF-15, and tumor metastatic potential, we performed
quantitative real-time PCR analysis in colorectal cancer cell
lines SW480 and SW620, which were from the same patient.
Compared with the SW480 primary clone, the SW620
that originated from the metastatic lymph node shows a
higher tumor metastatic potential.
43,44
Consistently, both
Calu-15 and GDF-15 mRNA levels were significantly
increased in SW620 cells (Figure 5g, P¼2.25 10
7
and
P¼5.13 10
14
, respectively), suggesting a significant
correlation among Calu-15, GDF-15, and tumor metastatic
potential.
Discussion
In this study, we found that the CALU gene encodes 13 novel
isoforms besides Calu-1 and Calu-2. Among them, only Calu-
15 shows nuclear localization. We demonstrate that Calu-15
is phosphorylated at Thr-73 by CK2, and then enters the
nucleus to promote the transcription of GDF-15 via binding to
its promoter, resulting in enhanced filopodia formation and cell
migration (Figure 6). Moreover, the entry of Calu-15 into the
nucleus is mediated by importin-aand Ran GTPase, whereas
its export from the nucleus is mediated by Crm1.
Figure 4 Calumenin (Calu)-15 promotes the transcription of growth differentiation factor-15 (GDF-15). (a) Quantitative real-time PCR analysis of the relative mRNA
expression levels of the indicated genes in the HeLa cells stably expressing Calu-15–EGFP (n¼3). ABHD2, a-/b-hydrolase domain containing protein 2; NELL2, neural
epidermal growth factor-like 2; ITPR1, inositol 1,4,5-triphosphate receptor, type 1; PML, promyelocytic leukemia; EGFP, as a control. Data are mean±S.E.M.; ***Po0.001
(unpaired two-tailed Student’s t-test). (b) Lysates of HEK293T cells expressing Calu-15–2 EGFP or 2 EGFP were immunoblotted (western blotting (WB)) with the
indicated antibodies. Right panel, the graph shows the relative protein level of GDF-15 (n¼4). Data are mean±S.E.M.; ***Po0.001 (unpaired two-tailed Student’s t-test).
(c) Analysis of GDF-15 promoter activity by dual-luciferase reporter assay. The left panel shows the schematic structures of plasmids with different lengths of GDF-15 promoter
followed by luciferase gene. The right graph indicates the relative luciferase activity in Calu-15–EGFP-overexpressing HeLa cells transfected with the indicated plasmids
(n¼4). EGFP, as a control. Data are mean±S.E.M.; **Po0.01, ***Po0.001 (unpaired two-tailed Student’s t-test). (d) Chromatin immunoprecipitation (ChIP) assay of HeLa
cells expressing Calu-15–EGFP or EGFP. (e) Quantification of the GDF-15 promoter activity in HeLa cells expressing Calu-15–2 EGFP wild type (WT), T73A, or T73E
(n¼3). Data are mean±S.E.M.; *Po0.05 (unpaired two-tailed Student’s t-test)
Calumenin-15 facilitates filopodia formation
H Feng et al
6
Cell Death and Disease
One gene can produce more than one isoform to function
either similarly or differently.
23
Our results show that the CALU
gene produces 15 isoforms in human cells by alternative
splicing, while 13 of them are first identified. Among these 15
isoforms, Calu 1–14 all possess an N-terminal signal peptide
and might localize in the lumen of the endomembrane system.
However, only Calu-15, lacking the N-terminal 151 aa,
shows nuclear accumulation and has novel functions as a
transcription regulator. Interestingly, the homologue gene of
Drosophila melanogaster,SCF, also encodes a nuclear
isoform DmSCF (DNA supercoiling factor) through alternative
splicing.
45
Therefore, it suggests that the production of a
nuclear isoform of the CALU gene is evolutionarily conserved
and has a significant physiological function.
In eukaryotic cells, nuclear import receptors such as
importin-aand -brecognize the nuclear localization signal
Figure 5 Calumenin (Calu)-15 promotes cell migration and facilitates filopodia formation in a growth differentiation factor-15 (GDF-15)-dependent pathway. (a) Wound
sizes of HeLa cells expressing Calu-15–EGFP or EGFP at the indicated time points (n¼3). Data are mean±S.E.M.; ***Po0.001 (unpaired two-tailed Student’s t-test).
(b) HeLa cells transfected with Calu-15–2EGFP or 2 EGFP were stained with Atto 565 phalloidinto show the localization and distribution of filamentous (F)-actin. Left panel,
representative pictures. Bar, 10 mm. Right panel, the graph shows the percentage of cells with filopodia (n¼3; 4100 cells per experiment). Data are mean±S.E.M.;
***Po0.001 (unpaired two-tailed Student’s t-test). (c) HeLa cells transfected with Calu-15–2 EGFP wild type (WT), T73A, or T73E were stained with Atto 565 phalloidin. Left
panel, representative pictures. Bar, 10 mm. Right panel, the graph shows the percentage of cells with filopodia (n¼3; 4100 cells per experiment). Data are mean±S.E.M.;
***Po0.001 (unpaired two-tailed Student’s t-test). (d) Conditioned medium collected from HEK293T cells transfected with Calu-15–2 EGFP or 2 EGFP were added into
the cultures of HeLa cells, and then the HeLa cells were stained with Atto 565 phalloidin. Left panel, representative pictures. Bar, 10 mm. Right panel, the graph shows the
percentage of cells with filopodia (n¼3; 4100 cells per experiment). Data are mean±S.E.M.; ***Po0.001 (unpaired two-tailed Student’s t-test). (e) At 48 h after GDF-15
shRNA transfection, the protein level of GDF-15–EGFP in HEK293T cells were examined by immunoblotting (western blotting (WB)). N.C., negative control; 88, 91, and 92
indicate GDF-15 shRNA with different target sequences; Pool, a mixture of GDF-15 shRNA 88, 91, and 92. (f) At 48 h after cotransfection with Calu-15–2 EGFP and GDF-15
shRNA pool (Pool, lower panels) or N.C. shRNA (upper panels), HeLa cells were stained with Atto 565 phalloidin. Left panel, representative pictures. Bar, 10 mm. Right panel,
the graph shows the percentage of cells with filopodia (n¼3; 4100 cells per experiment). Data are mean±S.E.M.; ***Po0.001 (unpaired two-tailed Student’s t-test).
(g) Quantitative real-time PCR analysis of the relative mRNA expression levels of Calu-15 and GDF-15 in SW480 and SW620 cancer cells (n¼5). Data are mean±S.E.M.;
***Po0.001 (unpaired two-tailed Student’s t-test). For b,c,dand f, to quantify the number of cells with filopodia, cells were scored positive when presenting at least 10
filopodia Z4mm in length
Calumenin-15 facilitates filopodia formation
H Feng et al
7
Cell Death and Disease
(NLS) of the proteins and then form an import complex with
them to enter the nucleus,
33
whereas exportins such as Crm1
recognize the nuclear export signal (NES) and form an export
complex to exit.
33,36
In our experiment, Calu-15 interacts with
both importin-aand Crm1. However, we have not identified
any classical NLS and NES in Calu-15, suggesting that there
may be other proteins to mediate its nuclear-cytoplasmic
shuttling, or that Calu-15 contains non-classical NLS and
NES. Besides, we show that the phosphorylation of Calu-15 at
Thr-73 by CK2 is indispensable for its nuclear localization,
which is similar to recent reports that nuclear-cytoplasmic
shuttling can be regulated by protein modification such as
phosphorylation and dephosphorylation.
38,39
CK2 has been
reported to regulate the nuclear export of S6K1 II,
46
but in our
study it acts as a regulator to facilitate the nuclear import of
Calu-15. However, we still do not know whether the
phosphorylation at Thr-73 facilitates the binding of Calu-15
directly to importin-aor to some other scaffold proteins. More
detailed mechanism needs further investigation.
GDF-15 functions as a cytokine, participating in various
cellular processes.
13–17
Meanwhile, it has pleiotropic func-
tions in cancer progression.
17
Because of its importance, the
transcription level of GDF-15 is under stringent control of
various regulatory pathways. Many transcription factors have
been reported to regulate its expression, such as p53, early
growth response protein 1 (EGR-1), Sp1, nuclear factor-kB,
poly (ADP-ribose) polymerase-1, and hypoxia-inducible
factor-1a.
14,47–51
Our results show that Calu-15, as a new
GDF-15 regulator, binds to the promoter region of GDF-15,
and our preliminary data suggests that other factors might be
required for this binding (data not shown). The binding region
of Calu-15 to GDF-15 we identified ranges from 168 to
þ32 bp around the transcription start site, in which it also
contains one p53- (at þ21 bp), three Sp1- (within the 133 to
þ41 bp), and two EGR-1- (within the 73 to 51 bp) binding
sites.
21,47,48
Whether Calu-15 functions coordinately with
them to promote the expression of GDF-15 remains unclear,
however, it is evident that this 200-bp region of GDF-15
promoter is crucial for its transcription. In addition, we do not
exclude the possibility that Calu-15 regulates the transcription
activities of other genes.
GDF-15 has been reported to promote filopodia formation
and metastasis of human prostate cancer cells through the
focal adhesion kinase (FAK)-RhoA signaling pathway.
13
Here we show that Calu-15 functionally promotes cell
migration by facilitating filopodia formation and this function
depends on GDF-15. Therefore, future research to investi-
gate whether Calu-15 also promotes filopodia formation
through FAK-RhoA signaling pathway could facilitate our
understanding of the detailed mechanism. Besides, the
function of GDF-15 in tumor progression is multiple but the
regulatory mechanism of GDF-15 in cancer cells is still
incompletely understood, whereas tumor cells with abundant
filopodia are identified to be more malignant and invasive
during tumor progression.
3–5
Considering the above two
facts, Calu-15, as a novel regulator of GDF-15, might have a
pro-tumorigenic role in cancer development and serve as a
new target for cancer therapy.
Materials and Methods
Cell lines and culture. HeLa, HEK293T, and SW480 cells were cultured at
37 1C with 5% CO
2
in Dulbecco’s modified Eagle’s medium (GIBCO BRL, Grand
Island, NY, USA) containing 10% heat-inactivated fetal bovine serum.
52
SW620
cells were cultured at 37 1C without CO
2
in Leibovitz’s L-15 medium (GIBCO BRL)
containing 10% heat-inactivated fetal bovine serum.
RNA isolation and cDNA cloning. Cells were homogenized in TRIzol
reagent (Invitrogen, Carlsbad, CA, USA) and the total RNA was isolated as
described in Molecular Cloning (third version). Subsequently, total RNA and
Superscript III Reverse Transcriptase (Invitrogen) were used to synthesize the
first-strand cDNA, according to the manufacturers’ instructions. The cDNAs
synthesized were then used for Calu cloning and quantitative real-time PCR
analysis. All PCR products were delivered for sequencing (Invitrogen).
Vector construction. To gain all the transcripts of Calu isoforms, specific
primers were synthesized and isoforms were cloned into the EcoRI and SalI site
of the pEGFP-N3 vector (Clontech Laboratories, Mountain View, CA, USA).
2EGFP plasmid was constructed by cloning EGFP into the KpnI and SmaI site
of the pEGFP-N3 vector. All the other mutants of Calu-15 were cloned into the
EcoRI and SalI site of the 2 EGFP vector. The promoter of GDF-15 was cloned
into the pGL3-Basic vector (Promega, Madison, WI, USA).
Antibodies. Mouse anti-calu polyclonal antibody was produced previously.
52
Rabbit anti-EGFP polyclonal antibody was produced in our lab and was used in
immunoblotting and immunoprecipitation. Rabbit anti-importin-a, rabbit anti-lamin-
A and -C polyclonal antibody, and mouse anti-Ran GTPase polyclonal antibody
were from Professor CM Zhang (Peking University). Mouse anti-a-tubulin
monoclonal antibody (DM1a) was from Sigma (St. Louis, MO, USA). Mouse
anti-GAPDH monoclonal antibody (ab8245) was from Abcam (Cambridge, UK).
Mouse anti-GFP monoclonal antibody (clone 1E4) was from MBL (Woburn, MA,
USA). Rabbit anti-GDF-15 monoclonal antibody (D2A3) was from Cell Signaling
Technology (Danvers, MA, USA).
Immunoblotting analysis. Immunoblotting analysis were preformed as
described.
52
Briefly, the proteins were transferred onto PVDF membranes
(Millipore, Billerica, MA, USA) in a semidry transfer cell (Bio-Rad, Hercules, CA,
USA) after gel separation by SDS-PAGE. Next, the membranes were blocked in
3–5% milk in TTBS for 30 min and probed with primary antibodies at 4 1C
Figure 6 Proposed working model of calumenin (Calu)-15. Calu-15 undergoes
phosphorylation at threonine at position 73 (Thr-73) by casein kinase 2 (CK2) and
then promotes its entry into the nucleus. In the nucleus, Calu-15 can activate the
transcription of growth differentiation factor-15 (GDF-15) via binding to its promoter
region, and then leads to the formation of filopodia, which in turn promotes cell
migration
Calumenin-15 facilitates filopodia formation
H Feng et al
8
Cell Death and Disease
overnight. HRP-conjugated goat anti-mouse or anti-rabbit IgG (Jackson
ImmunoResearch Laboratories, West Grove, PA, USA) were used as secondary
antibodies. To test the phosphorylation, SDS-PAGE with 50 mM Phos-tag
acrylamide AAL-107 (Wako Pure Chemical Industries, Osaka, Japan) was used.
Transfection, RNAi, and immunofluorescence. HeLa cells were
transiently transfected by jetPEI (Polyplus, Illkirch, France) at B60% confluence
following the manufacturer’s instructions. At 24 h post transfection, cells were
washed in PBS and then fixed with freshly prepared 4% PFA (paraformaldehyde)
in PBS for 15 min at 37 1C. Subsequently, cells were permeabilized with 0.1%
Triton X-100 in PBS buffer for another 15 min and blocked by 3% BSA in PBS for
30 min. Next, they were incubated with primary and secondary antibodies or Atto
565 phalloidin (Sigma), and observed under a fluorescence microscope (Olympus,
Shinjuku, Tokyo, Japan) as previously described.
53
For GDF-15 knockdown, cells
were transfected with plasmids expressing short hairpin RNA targeting GDF-15
(88: 50-GCTCCAGACCTATGATGACTT-30; 91: 50-CCGGATACTCACGCCAGAA
GT-30; and 92: 50-CTATGATGACTTGTTAGCCAA-30; Sigma), then they were
lysed for western blotting or fixed for immunofluorescence at 48 h post
transfection.
Immunoprecipitation assay. For the immunoprecipitation assay,
HEK293T cells transfected with Calu-15–EGFP were lysed in the buffer (20 mM
Tris-HCl, 150 mM NaCl, 0.1% NP-40, 1 mM DTT, 0.5mM EGTA, 10% glycerol,
1 mM MgCl
2
, pH 7.9) containing a protease inhibitor cocktail (Roche, Basel,
Switzerland). Next, the total cell lysates were incubated with anti-EGFP antibody at
41C overnight. The protein samples that combined with the Protein A Sepharose
(GE Healthcare, Uppsala, Sweden) were collected and subjected to western
blotting assay.
Nuclear and cytoplasmic extraction. HeLa cells were first incubated
with buffer 1 (20 mM HEPES, 5 mM CH
3
COOK, 0.5 mM MgCl
2
, 1 mM DTT, pH
7.8) on ice for 10 min and then homogenized by 25 strokes of a glass Dounce
homogenizer. The cytoplasmic fraction was gained by first centrifugation at
4000 r.p.m. for 5 min and second centrifugation of the supernatant at 20000 g
for 30 min. The fraction remaining from the first centrifugation was incubated with
buffer 2 (20 mM HEPES, 5 mM CH
3
COOK, 0.5 mM MgCl
2
, 1 mM DTT, 0.4 M
NaCl, pH 7.8) at 4 1C for 9 min and then centrifuged at 14 000 r.p.m. for 30 min to
gain the nuclear fraction. Nuclear and cytoplasmic fractions were then subjected to
western blotting.
Quantitative real-time PCR. The ABI 7300 Detection System (Applied
Biosystems, Foster City, CA, USA) was used to perform quantitative real-time
PCR using the SYBR Green PCR Master Mix (Applied Biosystems) as previously
described.
54,55
The primers used for quantitative real-time PCR were listed in
Supplementary Table S1. GAPDH served as a reference control and the 2
DDCT
method was used.
56
Dual-luciferase reporter assay. Different lengths of the promoter region
of GDF-15 were amplified from genomic DNA and cloned into the pGL3-Basic
vector. Next, 50 ng of the pGL3-Basic vectors containing different lengths of GDF-
15 promoter were transfected into HeLa cells, together with the indicated plasmids
and 50 ng of TK-Renilla receptor gene as an internal control. After 24 h, the
luciferase activity of total cell lysates was tested by the Dual-Luciferase Reporter
Assay System (Promega).
Chromatin immunoprecipitation assay. ChIP assay was performed as
described.
57
Briefly, cells were cross-linked by adding a final concentration of 1%
formaldehyde for 10 min at room temperature and cross-linking was stopped by
adding glycine to a final concentration of 125 mM. Cells were then washed with
1PBS, resuspended in cell lysis buffer (5 mM Pipes (KOH), 85 mM KCl, 0.5%
NP-40, pH 8.0), and centrifuged to get the nuclei. The nuclear fraction was then
resuspended in nuclear lysis buffer (50 mM Tris, 10 mM EDTA, 1% SDS, pH 8.1),
sonicated, and centrifuged to get the supernatant containing chromatin shorter
than 500 bp. The supernatant was diluted five-fold in ChIP dilution buffer (0.01%
SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris, 167 mM NaCl, pH 8.1) and
incubated with anti-EGFP antibody at 4 1C overnight. Protein A Sepharose beads
(GE Healthcare) were used to collect the immune complex and then washed
consecutively for 3–5 min with 1 ml of each solution: low-salt wash buffer (0.1%
SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris, 150 mM NaCl, pH 8.1), high-salt
wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20mM Tris, 500 mM NaCl,
pH 8.1), and LiCl wash buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM
EDTA, 10 mM Tris, pH 8.0), twice in 1 TE buffer (10 mM Tris, 1 mM EDTA,
pH 8.0). The complexes were eluted by adding elution buffer (1% SDS, 0.1 M
NaHCO
3
). Next, the formaldehyde cross-linking in the elutant was reversed and
the DNA was purified by a Gel Extraction Kit (CWBIO, Beijing, China). The purified
DNA fraction was then analyzed by PCR using appropriate primers.
Wound-healing assay. HeLa cells stably expressing EGFP or Calu-15–
EGFP were planted on coverslips at the same density. A micropipette tip was used
to scratch a wound. The detached cells were removed and the remaining cells
were cultured at 37 1C. The wound sizes at the same site were recorded as the
indicated time.
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgements. We thank Professor WG Zhu from the Peking University
for SW480 cell line, Professor CM Zhang from the Peking University for the rabbit
anti-importin-a, and rabbit anti-lamin-A and -C polyclonal antibody and mouse anti-
Ran GTPase polyclonal antibody, and Professor IC Bruce 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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Calumenin-15 facilitates filopodia formation
H Feng et al
10
Cell Death and Disease
