{'fraction_non_alphanumeric': 0.04214927668573076, 'fraction_numerical': 0.03417119238717739, 'mean_word_length': 4.958762886597938, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Calumenin-15 facilitates filopodia formation by promoting TGF-β superfamily cytokine GDF-15 transcription

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 α, 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-β (TGF-β) 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.


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][2][3] whereas abundant filopodia are considered strongly correlated with the enhanced cell migration and tumor metastasis. [3][4][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][7][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-b superfamily 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-b superfamily 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][14][15][16][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 localizations 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 (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 2 þ -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).
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 Â EGFP and found that Calu-15-2 Â 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 Â 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 pathway 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 complex 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 Â 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 phosphorylation 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 (N ¼ C);  Figure 3a, P ¼ 1.41 Â 10 À 33 and P ¼ 0.10, respectively). Thus, these results suggest that the phosphorylation of Calu-15 at Thr-73 is related to its nuclear localization.
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-EGFPexpressing cells (Figure 4c 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, P ¼ 0.0337). 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-15mediated pathway. Interestingly, we found that overexpression of Calu-15-EGFP promoted cell migration in the woundhealing assay (Figure 5a and Supplementary Figure S4). Besides, Calu-15 also significantly increased the number and size of filopodia (Figure 5b, P ¼ 9.59 Â 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, P ¼ 1.46 Â 10 -5 , P ¼ 5.03 Â 10 À 6 and P ¼ 3.84 Â 10 À 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 Â EGFP-expressing HEK293T cells and the filopodia formation was also induced (Figure 5d 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, P ¼ 2.25 Â 10 À 7 and P ¼ 5.13 Â 10 À 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-a and Ran GTPase, whereas its export from the nucleus is mediated by Crm1. 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-a and -b recognize the nuclear localization signal (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-a and 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-a or to some other scaffold proteins. More detailed mechanism needs further investigation.
GDF-15 functions as a cytokine, participating in various cellular processes. [13][14][15][16][17] Meanwhile, it has pleiotropic functions 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][48][49][50][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 À 168 to þ 32 bp around the transcription start site, in which it also contains one p53-(at þ 21 bp), three Sp1-(within the À 133 to þ 41 bp), and two EGR-1-(within the À 73 to À 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 investigate 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][4][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.
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). 2 Â EGFP 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 Â 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  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 À 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).
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.