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1 Department of Cardiology, Cardiovascular Research Institute, Shenyang Northern Hospital, Shenyang 110016, China
2 Department of Surgery, Division of Vascular Surgery, Robert Wood Johnson Medical School-UMDNJ, NJ 08903, USA
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Abstract |
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Introduction |
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Our previous studies showed that CREG is significantly up-regulated at both the mRNA and protein levels during phenotypic conversion of proliferative and synthetic smooth muscle cells (SMCs) to non-proliferative and differentiated SMCs in vitro (Han et al. 2005). Moreover, CREG over-expression in cultured SMCs may inhibit cellular proliferation and promote differentiation, whereas CREG knockdown prevents serum starvation-induced SMCs maturation and growth arrest. Furthermore, CREG is down-regulated in the vascular media after balloon injury to the rabbit carotid artery. Adenovirus-mediated CREG over-expression in injured arteries inhibits SMCs proliferation and attenuates neointimal hyperplasia in vivo (Wang et al. 2008).
Research therefore suggests that CREG plays a critical role in keeping cell or tissue in a mature, homeostatic state by antagonizing pathological de-differentiation and overgrowth. It is therefore of interest to further defining the role of CREG in regulating cell growth and to identify a potential cell surface receptor for secreted CREG protein.
The human homolog of CREG contains 220 amino acid residues and 3-consensus N-glycosylation sites (Sacher et al. 2005). A functional signal sequence at its amino terminus targets the CREG protein into the endoplasmic reticulum and shows its localization in the perinuclear region, a typical site for secreted proteins. It has been proposed that CREG acts as an extracellular ligand binding to a cell surface receptor. Of the several proteins that had been reported to interact with CREG, the cation-independent mannose-6-phosphate (M6P)/ insulin-like growth factor II receptor (IGF2R) has been shown to be required for its growth-suppressive activity (Di Bacco & Gill 2003). M6P/IGF2R is a multifunctional transmembrane glycoprotein of 300 kDa, with a large extracellular domain containing 15 repeat regions and a small cytoplasmic domain. A major function of the receptor is to bind and transport M6P-bearing glycoproteins (e.g., lysosomal enzymes) from the trans-Golgi network or cell surface to lysosomes (De Souza et al. 1997; Kang et al. 1998). Cell surface M6P/IGF2R also binds and internalizes insulin-like growth factor II (IGF-II), resulting in the lysosomal degradation of this ligand (Oka et al. 1985). In this manner, the receptor may serve as a suppressor of IGF-II's proliferative actions. In addition, M6P/IGF2R binds latent transforming growth factor-β, permitting cleavage into its active form, which is a potent growth inhibitor for most cell types (Dennis & Rifkin 1991). A mutation of M6P/IGF2R is often found in many kinds of carcinoma cells (Tsujiuchi et al. 2004; Hu et al. 2006; Iwamoto et al. 2006), this suggests that it may help to inhibit pathological cellular proliferation and that it therefore plays a similar role to that of CREG in inhibiting cell overgrowth. If so, understanding how CREG and M6P/IGF2R cooperate will shed light on its role in carcinogenesis, embryonic development, and the pathogenesis of atherosclerosis.
In the present study, we show for the first time that forced knockdown of the expression of CREG promotes the cellular proliferation associated with the increase of the IGF-II in NIH3T3 fibroblasts. Moreover, the effect of CREG knockdown on cell proliferation is markedly inhibited in a concentration-dependent manner by re-addition of recombinant CREG protein into the media of cultured cells, and this was mediated by membrane receptor M6P/IGF2R. Although the direct interactions between CREG and M6P/IGF2R were identified by both immunoprecipitation (IP)-Western blotting and immunofluorescence staining, subsequent analysis of the expression and localization of the receptor suggests that CREG knockdown remarkably attenuates the localization of the receptor in the cytoplasm and cell membrane but does not affect its expression in NIH3T3 fibroblasts. This re-distribution of M6P/IGF2R onto cellular membrane requires further investigation.
Our data indicates that secreted CREG may act as the functional regulator of M6P/IGF2R to facilitate binding and trafficking of IGF-II endocytosis, leading to growth inhibition.
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Results |
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Previous studies, including our own, have shown that CREG expression closely correlates with cell proliferation and differentiation and that over-expression leads to cellular differentiation and growth suppression in many types of cells (Veal et al. 2000; Di Bacco & Gill 2003; Han et al. 2005). To confirm the biological effects of CREG on cell proliferation, our current study first generated stable NIH3T3 fibroblasts that knocked down the expression of CREG by transfection of pDS_shCREGs vectors. As shown in Fig. 1A, stable transfection of shCREG1 vector into NIH3T3 fibroblasts led to CREG expression of at most approximately 80% decrease of that of the control plasmid transfected cells and/or normal cultured cells, either in the lysates or in the medium (P < 0.01).
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Recombinant hCREG protein inhibits cell growth mediated by M6P/IGF2R
To further determine the effects of CREG expression on cell proliferation, we constructed an eukaryotic vector that expressed recombinant human CREG (hCREG) with the myc-his tagged at C-terminal and transfected it into human 293 cells to produce recombinant hCREG/myc-his protein. We used a Ni-NTA agarose affinity chromatography column to purify recombinant hCREG/myc-his protein from the lysates of 293 cells with stable transfection. When the recombinant hCREG was added to the medium, the cell proliferation induced by CREG knockdown was remarkably inhibited in a concentration-depended manner, as assessed by both FACS and BrdU assay (Fig. 2A,B). The results show that recombinant hCREG is a functional protein with ability on growth-inhibition. This is consistent with previous reports that secreted CREG significantly inhibits cell proliferation possibly mediated by the specific membrane receptor protein.
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Current studies (Di Bacco & Gill 2003; Sacher et al. 2005; Jeanjean et al. 2006) suggest that M6P/IGF2R plays a critical role as a negative regulator of cell growth, similar to CREG. We therefore investigated whether M6P/IGF2R is involved in CREG's regulating growth inhibition of NIH3T3 fibroblasts. When various dilutions of the anti-mouse IGF2R neutralization antibody were used to block the activity of M6P/IGF2R, FACS analysis showed that all dilutions (1 : 20 to 1 : 200) of IGF2R antiserum abrogated the effects of CREG on cell growth inhibition. The greatest inhibition of the effects of CREG occurred at a 1 : 50 dilution (Fig. 2E). This demonstrates that the effects of CREG on growth inhibition of fibroblasts may be mediated by membrane protein M6P/IGF2R binding directly to CREG.
CREG regulates M6P/IGF2R localization but does not affect its expression
Although we identified that M6P/IGF2R is involved in the regulation of CREG's effects on cell proliferation, the mechanisms behind these interactions remained unknown. We therefore used Western blot and immunofluorescence staining to investigate the expression and distribution of the receptor in NIH3T3 fibroblasts lacking or expressing CREG. Western blotting showed that there was no significant difference in the expression of M6P/IGF2R in NIH3T3 fibroblasts lacking CREG compared to those expressing CREG (Fig. 3A). Whereas the immunofluorescence staining revealed a remarkable difference M6P/IGF2R distribution in NIH3T3 fibroblasts depending on whether they lacked or expressed CREG. As illustrated in Fig. 3B, we investigated the localization of M6P/IGF2R both in the perinuclear structure and in the cell membrane in normal cultured NIH3T3 where M6P/IGF2R was abundant and found it was primarily concentrated in the perinuclear structure (probably the TGN), cytoplasm (probably the endosome and lysosome), and cell membrane. In contrast to NIH3T3 fibroblasts lacking CREG, cells with CREG had a dramatically altered distribution of the receptor, which was found only in the perinuclear structure. Additionally, when recombinant hCREG protein was added into the media of cells lacking CREG, the receptor was re-distributed to the cytoplasm and cell membrane. These observations strongly indicate that expression of CREG plays a critical role in the distribution and trafficking of M6P/IGF2R, although it does not alter its expression.
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The M6P/IGF2R is well-characterized as a trafficking receptor, the activities of which include intracellular transportation to lysosomes, localization of proteins at the cell surface, and internalization of extracellular ligands. Among these functions, binding and internalizing the extracellular secreted IGF-II, resulting in the lysosomal degradation of this ligand, is one of the most important. The receptor may serve as a suppressor of IGF-II's proliferative actions (Kang et al. 1998). Our previous studies identified that reduction of CREG attenuates the localization of M6P/IGF2R in the cell membrane. Therefore, we hypothesize that the CREG may be involved in regulating IGF-II endocytosis and degradation mediated by M6P/IGF2R. In the present study, we used ELISA to detect secreted IGF-II in the media of NIH3T3 fibroblasts, which did or did not express CREG. Compared to levels in cells expressing CREG, we found that reducing CREG expression markedly increased levels of IGF-II (P < 0.01) in NIH3T3 fibroblasts. Conversely, adding recombinant hCREG protein abrogated the up-regulation of IGF-II stimulated by CREG knockdown (Fig. 4A). Furthermore, when the anti-mouse IGF-II neutralize antibody was treated into the medium to bind the IGF-II, the effect of cellular proliferation was identified to block obviously both by FACS analysis and BrdU assay (Fig. 4B,C).
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Discussion |
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This is the first study using mouse CREG siRNA vector to forcibly inhibit expression of CREG in NIH3T3 fibroblasts. We clearly found that CREG knockdown promotes cell proliferation. Furthermore, when recombinant hCREG protein is added to the media of the cells, the cell proliferation induced by CREG knockdown is significantly inhibited in a concentration-dependent manner. In consistent with previous reports, we also found that reducing CREG may be an early step in promoting pathologic proliferation in mature cells (Gordon et al. 2005). Our work uses a different cell lineage to expand current knowledge of the role of CREG and provides genetic evidence that CREG is a major regulator of cell growth and tissue differentiation. Further, we confirm the ability of recombinant CREG protein to antagonize cell proliferation: this may be utilized for therapeutic benefit in some proliferative disease, such as some human cancers and atherosclerosis.
To clarity the transduction pathway by which CREG protein inhibits fibroblasts proliferation and to determine the cell surface receptor involved, we assessed whether CREG and its putative membrane receptor, M6P/IGF2R, physically interact in NIH3T3 fibroblasts. First, using IP-Western blotting and immunofluorescence staining analysis, we showed that CREG can bind directly to M6P/IGF2R in NIH3T3 fibroblasts, although the exact sites of their interactions remain unclear. Subsequently, we added a neutralizing antibody (anti-M6P/IGF2R) to the medium of NIH3T3 fibroblasts and co-incubated this with recombinant CREG protein. We found that CREG's ability to inhibit cell growth was completely blocked when the antibody is diluted to 1 : 50. This suggests that the ability of exogenous CREG to inhibit cell proliferation is mediated by M6P/IGF2R.
M6P/IGF2R is a multifunctional transmembrane glycoprotein. As with CREG, M6P/IGF2R is essential for normal fetal development and cell growth. Loss of function resulting from mutations or deletions of this receptor are associated with human tumors in a variety of tissues including liver, breast, lung, head and neck, and gastrointestines (Oates et al. 1998; Kong et al. 2000; Jamieson et al. 2003). M6P/IGF2R may function as a growth and tumor suppressor by regulating the localization, activity, and degradation of many ligands including both M6P-bearing proteins and non-glycosylated proteins (Faust et al. 1987; Lee & Nathans 1988; Blanchard et al. 1998). Therefore, we hypothesize that secreted CREG protein may be one of the ligands involved in regulating M6P/IGF2R's biological functions.
In this study, we investigated the expression and localization of M6P/IGF2R in cells either lacking or expressing CREG, using Western blotting or immunofluorescence staining. Although Western blot analysis shows that there is no significant difference in the levels of M6P/IGF2R expression, immunofluorescence staining reveals a remarkable M6P/IGF2R distribution in NIH3T3 fibroblasts lacking CREG. In cultured NIH3T3 fibroblasts expressing CREG, M6P/IGF2R is primarily concentrated in the perinuclear structure (probably the TGN), cytoplasm (probably the endosome and lysosome), and cell membrane. However, in NIH3T3 fibroblasts lacking CREG, the distribution of the receptor is dramatically different, being localized only in the perinuclear structure. Furthermore, adding recombinant hCREG protein to the media of NIH3T3 fibroblasts causes the receptor to be re-distributed into the cytoplasm and cell membrane. These observations strongly indicate that the CREG protein plays a critical role in regulating the distribution and trafficking of M6P/IGF2R, although it does not alter its expression.
One of the best-characterized functions of M6P/IGF2R is to mediate the endocytosis and degradation of extracellular IGF-II in lysosomes, thereby suppressing mitogenesis by reducing IGF-II availability for binding to the IGF-I receptor (Kawamoto et al. 1998). Loss of imprinting of the M6P/IGF2R gene, which may be responsible for increasing IGF-II mRNA and protein production, occurs in sporadic Wilms tumors (Xu et al. 1997), lung carcinomas (Tsujiuchi et al. 2003), and human prostate tissue (Hu et al. 2006). Additionally, positive staining for IGF-II correlates with increased tumor progression, increased proliferating cell nuclear antigen staining, and decreased patient survival in colorectal cancers (Wang et al. 2005; Maenaka et al. 2006). This suggests that M6P/IGF2R plays a role in cancer growth suppression, possibly through internalization and degradation of IGF-II, which is a potent mitogen for many cells. Therefore, an important question raised by the interaction of CREG with M6P/IGF2R localization is whether the CREG involves the degradative pathway of IGF-II.
In this study, we detected secreted IGF-II in the media of M6P/IGF2R either lacking or expressing CREG, using ELISA. Reducing CREG expression markedly increased levels of IGF-II (P < 0.01) in the medium of NIH3T3 fibroblasts, and adding recombinant hCREG protein abrogated the up-regulation of IGF-II stimulated by CREG knockdown. Furthermore, when anti-mouse IGF-II neutralizing antibody was added to the medium to bind IGF-II, cell proliferation decreased. These findings indicate that the interaction between CREG and M6P/IGF2R is functionally important in inhibiting fibroblasts proliferation, which involves regulating translocation of M6P/IGF2R and altering the IGF-II endocytosis. However, the mechanism by which the binding of CREG to M6P/IGF2R regulates the translocation and internalization of the receptor remains unknown.
Our findings indicate that CREG protein may be a target for cell growth inhibition mediated by attenuation of IGF-II-induced endocytosis of its membrane receptor, M6P/IGF2R. Delivering recombinant CREG protein in vivo may provide therapeutic benefits for some pathological proliferative disease, such as arterial restenosis after angioplasty, atherosclerosis, and human cancers.
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Experimental procedures |
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NIH3T3 fibroblasts were obtained from ATCC (CRL-1658TM) and were grown in Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 4 mmol/L glutamine, 100 U/mL penicillin, and 100 U/mL streptomycin (complete medium).
Construction of RNAi vectors and generation of stable cells with CREG knockdown
Mouse CREG (Gene bank accession number NM_11804) mRNA target sequences were designed using a short interference RNA (siRNA) template design tool (http://www.dharmacon.com). The CREG mRNA target sequences were: CREG1:5'-CAAGACAGAAGAGGACTAT-3'; CREG2 5'-TCGCGGA CATCATCTCAAT-3', CREG3: 5'-CTGGCCACTATCTC CACAATA-3', CREG4: 5'-GAATGAAT CCTTCAGTCGAGG-3'. One non-sense oligonucleotide RNA fragment contained a 14 bp-nucleotide: 5'-TCGAGGCGGCCGCG-3' as a control. The double-strained oligonucleotides were cloned into the entry vector pEN_mh1c by BamHI and XhoI. Subsequently, the recombinant vectors were subcloned respectively into the short hairpin RNA (shRNA) expression vector pDS_hpEy to construct the pDS_shCREG and pDS_control vectors using the LR recombinant kit. All the recombinant plasmids were confirmed by DNA sequencing. The NIH3T3 fibroblasts were transfected with recombinant plasmids expressing CREG shRNAs, and control shRNA using LipofectamineTM 2000 reagent (Invitrogen) and were selected with 500 µg/mL G418. CREG knockdown was evaluated by Western blot analysis with an anti-CREG antibody (R&D Systems, Minneapolis, MN). The irrelevant β-actin served as a control.
Expression and purification of recombinant CREG
The recombinant CREG expression vector, designated pcDNA3.1-myc-His/hCREG, was constructed and contained the amino acid residues from 1 to 220. The recombinant vector was transferred into a human embryonic kidney cell line (293T) and the cell clones stabled transfer were obtained by selection with 500 µg/mL G418. Cells were harvested and the cell pellets stored at –20 °C. Frozen cells were thawed and re-suspended in lysis buffer (50 mM Tris, pH 7.5, 400 mM NaCl, 20 mM imidazole, 5 mM 2-mercaptoethanol, 5% glycerol, 1% Triton X-100, one tablet of protease inhibitor mixture without EDTA, 30 mL of buffer) followed by sonication on ice.
The lysate was centrifuged at 14 000 g. The supernatant was combined with 2 mL of a nickel (II)–nitrilotriacetic acid (Ni–NTA) agarose resin equilibrated in lysis buffer, and binding took place on a rotating wheel at room temperature, after which the entire content was poured into a PolyPrep column. The resin was washed successively with wash buffer I (lysis buffer without protease inhibitor and with 50 mM Tris, pH 8.0), wash buffer II (wash buffer I with 1 M NaCl and 40 mM imidazole), and wash buffer III (wash buffer II without Triton X-100 and with 400 mM NaCl). Proteins were eluted with elution buffer (200 mM imidazole, 50 mM Tris, pH 8.0, 200 mM NaCl, 5 mM 2-mercaptoethanol, 5% glycerol) for 10 min. The eluted protein was concentrated, and the buffer was exchanged for 150 mM NaCl, 50 mM Tris, and pH 7.5, 1 mM 2-mercaptoethanol. The final protein solution was concentrated to 1.6 mg/mL.
Immunoprecipitation and Western blot analysis
For IP, NIH3T3 were disrupted in lysis buffer (1% NP-40, 50 mM Tris, pH 7.4, 100 mM NaCl plus Sigma protease inhibitor cocktail) and incubated with an anti-CREG monoclonal antibody followed by protein G agarose beads. The IP was then analyzed by Western blotting. For Western blotting, NIH3T3 fibroblasts lysates was prepared in lysis buffer containing 1% SDS-PAGE gels. Proteins were resolved in SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. The primary antibodies used were anti-CREG, anti-IGF2R, and anti-β-actin antibodies. The specific binding was detected with HRP conjugated secondary antibodies and the ECL kit. The blots were quantified on a Bio-Rad gel documentation system.
Cell cycle and BrdU analysis
Cell cycle analysis was carried out in NIH3T3 fibroblasts serum-starved for 72 h and then stimulated with medium containing 10% serum for 36 h. After ethanol fixation and propidium iodide staining, NIH3T3 fibroblasts in distinct cell cycles were analyzed by fluorescence activated cell sorting (FACS Calibur, BD Biosiences). In order to identify replicating cells, we performed a BrdU incorporation assay with a cell proliferation kit (GE Healthcare Life Sciences). Cultured NIH3T3 fibroblasts were incubated with BrdU for 3-h before fixation. Incorporated BrdU was detected immunohistochemically with an anti-BrdU antibody.
IGF-II immunoassay
The level of secreted IGF-II in cell culture supernatants was measured using a commercial mouse IGF-II enzyme linked immunosorbent assay (ELISA) kit from R&D Systems. The immunoassay was performed using biological triplicates for samples (NIH3T3 fibroblasts culture treated with IGF-II) and controls (NIH3T3 fibroblasts left untreated). In addition, each biological sample was measured in duplicate. In order to compensate for the possibility that differences in cell number affected the amount of secreted IGF-II, the cells were counted and the final results are given as the mean amount of IGF-II produced per 106 cells.
Immunofluorescence analysis
NIH3T3 fibroblasts were plated overnight to achieve a 5%–10% confluent monolayer, then fixed with 4% paraformaldehyde and permeated with 0.2% Triton-X100, each for 15 min. After washing with PBS three times, nonspecific binding sites were blocked with 10% goat serum. The primary antibody was applied for overnight at 4 °C, followed by incubation with a tetramethyl rhodamine isothiocyanate (TRITC)-conjugated for IGF2R and fluorescein isothiocyanate (FITC)-conjugated for CREG secondary antibody (Santa Cruz Biotechnology) for 2 h at room temperature. The double staining sections were examined and photographed by confocal microscopy (OLYMPUS FV1000).
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Acknowledgements |
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Footnotes |
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* Correspondence: hanyaling{at}263.net
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References |
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Received: 27 April 2008
Accepted: 19 June 2008
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