Surface expression of heterogeneous nuclear RNA binding protein M4 on Kupffer cell relates to its...

Surface expression of heterogeneous nuclear RNA binding protein M4on Kupffer cell relates to its function as a carcinoembryonic antigen

receptor

Olga Bajenova,a,* Eugenia Stolper,a Svetlana Gapon,b Natalia Sundina,c

Regis Zimmer,a and Peter Thomasa

a Department of Surgery, Boston University School of Medicine, 801 Albany Street, Boston, MA 02118, USAb Department of Cardiology, Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA

c Massachusetts Institute of Technology, DCM Building 16, 77 Massachusetts Avenue, Cambridge, MA 02139, USA

Received 9 April 2003

Abstract

Elevated concentrations of carcinoembryonic antigen (CEA) in the blood are associated with the development of hepatic metastases fromcolorectal cancers. Clearance of circulating CEA occurs through endocytosis by liver macrophages, Kupffer cells. Previously we identifiedheterogeneous nuclear ribonucleoproteins M4 (hnRNP M4) as a receptor (CEAR) for CEA. HnRNP M4 has two isoform proteins (p80, p76),the full-length hnRNP M4 (CEARL) and a truncated form (CEARS) with a deletion of 39 amino acids between RNA binding domains 1and 2, generated by alternative splicing. The present study was undertaken to clarify any isoform-specific differences in terms of theirfunction as CEA receptor and localization. We develop anti-CEAR isoform-specific antibodies and show that both CEAR splicing isoformsare expressed on the surface of Kupffer cells and can function as CEA receptor. Alternatively, in P388D1 macrophages CEARS protein hasnuclear and CEARL has cytoplasmic localization. In MIP101 colon cancer and HeLa cells the CEARS protein is localized to the nucleusand CEARL to the cytoplasm. These findings imply that different functions are assigned to CEAR isoforms depending on the cell type. Thesearch of 39 amino acids deleted region against the Prosite data base revealed the presence of N-myristylation signal PGGPGMITIP thatmay be involved in protein targeting to the plasma membrane. Overall, this report demonstrates that the cellular distribution, level ofexpression, and relative amount of CEARL and CEARS isoforms determine specificity for CEA binding and the expression of alternativespliced forms of CEAR is regulated in a tissue-specific manner.© 2003 Elsevier Inc. All rights reserved.

Keywords: Carcinoembryonic antigen; Kupffer cells; Receptor; Macrophage; Heterogeneous RNA binding protein M4

Introduction

Carcinoembryonic antigen (CEA)1 is a cell surface gly-coprotein that is often overexpressed and secreted by colo-rectal and other tumors. It is a member of a large family of

related proteins and these all belong to the larger immuno-globulin supergene family [1]. Increasing levels of CEA inthe serum often correlate with the development of metasta-ses after surgical removal of primary colorectal tumors.CEA is widely used as a serum marker for colorectal cancerand for tumor-specific targeting [2,3]. The liver is the mostcommon site for metastasis from colorectal cancers with thelung as a secondary site [4]. Laboratory studies show arelationship between serum levels of CEA and the develop-ment of liver metastasis from colorectal carcinoma cells inan animal model [5]. In athymic nude mice, systemic injec-tion of CEA enhanced experimental liver metastasis andimplantation in the liver by weakly metastatic colorectal

* Corresponding author. Boston University School of Medicine, Lab-oratory of Surgical Biology, 801 Albany Street, Room 308, Boston, MA02118, USA. Fax: �1-617-414-8078.

E-mail address: [email protected] (O. Bajenova).1 Abbreviations used: KC, Kupffer cell; CEA, carcinoembryonic anti-

gen; hnRNP, heterogeneous RNA binding protein; RBD, RNA-bindingdomain

R

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Experimental Cell Research 291 (2003) 228–241 www.elsevier.com/locate/yexcr

0014-4827/$ – see front matter © 2003 Elsevier Inc. All rights reserved.doi:10.1016/S0014-4827(03)00373-2

cancer cell lines [6,7]. These weakly metastatic colon can-cer cell lines become highly metastatic when transfectedwith the cDNA coding for CEA [8,9]. We have shown thatCEA is rapidly cleared from the circulation of experimentalanimals, accumulates in the liver, and is endocytosed by areceptor (CEAR) on the liver fixed macrophages (Kupffercells) [10]. CEA binding is via a 5-amino-acid peptidelocated at the junction of the N-terminal and first (A1)immunoglobulin loop domain [11] and mutations in theregion of the gene coding for this sequence result in patientshaving grossly elevated serum CEA levels [12]. In isolatedKupffer cells CEA binding initiates a series of signalingevents that leads to phosphorylation on tyrosine in at leasttwo intracellular proteins [13]. These events are followed byinduction of IL-1�, IL-6, IL-10, and TNF-� cytokines [14].CEA can also form homotypic adhesion complexes that areinvolved in intercellular recognition [15]. However, CEA-induced hepatic metastasis seems to be associated withactivation of Kupffer cells and production of cytokines thatboth enhance adhesion of tumor cells to the hepatic endo-thelium [16] and protect against cytotoxicity by endothelialcells [17].

We cloned and sequenced the gene for the rat Kupffercell CEA receptor (CEAR) and identified it as an orthologof the human heterogeneous nuclear RNA binding proteinM4 (hnRNP M4) [18]. The hnRNP M4 protein belongs tothe subfamily of ubiquitously expressed heterogeneous nu-clear ribonucleoproteins (hnRNPs). These proteins aresometimes referred as the “histones of RNA” and are amongthe most abundant proteins in the metazoan nucleus [19].They are associated with pre-mRNAs and appear to influ-ence pre-mRNA processing and other aspects of mRNAmetabolism and transport. While all of the hnRNPs arepresent in the nucleus, some seem to shuttle between thenucleus and the cytoplasm [20]. Shuttling hnRNPs not onlyare involved in nuclear functions, but also play other cellu-lar roles, such as the transport of mature RNAs to thecytoplasm, mRNA translation, regulation of mRNA stabil-ity [21], and cell signaling [22–25]. The HnRNP M proteinswere first isolated as a group of closely related proteins,which run on two-dimensional SDS–PAGE gel as a clusterof four splicing isoforms, M1 to M4 [26]. Interestingly, acDNA clone for one of these proteins expressed in a rabbitreticulocyte lysate system selects polyadenylated RNA thattranslates into all four M proteins and this indicates that Mproteins are closely related to each other [27]. Alternativelyspliced transcripts for hnRNP M4 have been described [28].The full-length human hnRNP M4, CEARL (long), wasassigned at GenBank as isoform “A”. The rat hnRNP M4protein with the deletion of 29 amino acids (187–226) in aregion between RNA binding domains 1 and 2 (RBD1–RBD2) was isolated as the CEA receptor CEARS (short)[18]. Several reports show that hnRNP M4 is involved inmRNA splicing [28,29] and stress responses [30]. By ap-plying specific immunoprecipitation assays in a consecutiveseries of splicing reactions, in vitro evidence of almost

exclusive association of hnRNP M4 with pre-mRNA withinthe prespliceosomal A complex was provided [28]. Due tothis unique regulatory function in the mechanism that turnssplicing on and off, hnRNP M4 protein appears to be im-portant for controlling gene expression. Moreover, afterstress treatment (heat shock) hnRNP M is recruited into asmall number of nuclear bodies termed either HAP bodiesor stress bodies [31]. The hnRNP M4 protein is therefore amultifunctional protein that has roles in what appear to beunrelated processes. However, there is no information aboutthe functional differences of hnRNP M4 protein isoforms.The elucidation of these multiple functions is important tothe understanding of CEA-related signaling mechanism.

Here, we demonstrate by using anti-CEAR isoform-spe-cific antibodies and RT-PCR the differences in the expres-sion and cellular localization between two CEAR splicingvariants. These data support the hypothesis that it is thelocalization and the function of CEAR isoforms that makeKupffer cells specific for CEA binding.

Experimental procedures

Isolation of rat Kupffer cells

Kupffer cells were isolated from the livers of male Spra-gue–Dawley rats according to our standard laboratory pro-tocol [11]. The rodents were fasted overnight. The liverswere isolated and perfused through the portal vein withcollagenase. Suspended cells were separated into parenchy-mal and nonparenchymal fractions by differential centrifu-gation. Kupffer cells were purified from the nonparenchy-mal cell fraction using a gradient of 17.5% metrizamide inGey’s balanced salt solution. The interface layer containingKupffer cells was isolated and washed in phosphate-buffedsaline. Further purification of the cells was achieved byattachment to plastic plates for 2 h. Generally, 3–5 � 108

cells are routinely isolated from one liver. A total of 1 � 108

Kupffer cells remain after the adhesion step. Kupffer cellswere identified by their ability to phagocytose 1-�m latexparticles and by staining for endogenous peroxidase activ-ity. The cells were �90% viable by trypan blue staining and85% phagocytic.

Cell lines

PH1 and U937 human lymphoblasts, CRL2192 rat alve-olar, IC21 mouse peritoneal, Raw 246.7 mouse monocytes,and P388D1 adherent and suspension grown mouse lym-phoid macrophage cell lines were obtained from ATCC.The MIP 101 colorectal cancer cell line was developed fromcolon carcinoma biopsy and MIP101 (clone 17), a CEAproducing cell line formed by transfection of MIP-101 withthe full-length human CEA cDNA [9]. P388D1 suspensioncells were propagated in DMEM medium supplemented

229O. Bajenova et al. / Experimental Cell Research 291 (2003) 228–241

with 10% horse serum. CRL 2192 cells were grown inF-12K medium (Kaighn’s modification) supplemented with15% FBS. PH1, U937, P388D1 adherent, IC21, Raw 246.7,MIP101, and MIP101 (clone 17) cells were grown in RPM11640 (ATCC modified medium) supplemented with 10%FBS. All cells were maintained at 37°C in a 5% CO2

atmosphere. All tissue culture products were obtained fromLife Technologies.

CEAR transient transfection into macrophage cell lines

Cells were plated at 2–2.5.105 cells per well in six-welltissue culture clusters (Nunc products) and allowed to growfor 24 h. Plasmid constructs were transfected in serum-freemedium using SuperFect transfection reagent according tothe Qiagen protocol. A total of 2 �g of CEAR cDNA inpGEX4T-3 expression vector was transfected per well. For-ty-eight hours posttransfection cells were harvested andresuspended in a volume of 200 �l for measurement of CEAbinding activity.

In vitro CEA uptake assay

These procedures were performed as described previ-ously [9]. Briefly, CEA (5 �g/ml) labeled with 125I wasincubated with the cells in duplicate at 37°C. Samples weretaken at 0-, 15-, 30-, 45-, and 60-min intervals. These wereimmediately centrifuged through an oil phase (3:1 ratio ofdibutylphthalate: dioctylphthalate) at 14,000 rpm using anEppendorf microfuge. The cells spin through the oil and canbe separated from the supernatant for � counting by cuttingthe bottom off the plastic centrifuge tubes and collecting thecell pellet.

Production of anti-CEAR antibodies specific to thesplicing isoforms

We developed three antibodies specific to either CEARS

or CEARL or both splicing variants using synthetic anti-genic peptides. The location of these peptides on the CEARsequence is presented in Fig. 1. As these regions of CEARprotein are identical in human and rat sequences, the anti-bodies will recognize both species.

1. The first antigenic peptide was designed for the anti-body to recognize the full-length protein (CEARL).The 22-amino-acid MATTGGMGMGPGGPGMIT-IPPS peptide corresponds to the part of the spacerregion between RBD1 and RBD2 on the N-terminusof the protein. The search of these peptide against theProsite data base revealed the presence of N-myristy-lation signal PGGPGMITIP that is involved in proteintargeting to the plasma membrane [32].

2. The second antigenic peptide was designed for theantibody to recognize CEARS isoform with the dele-tion in a spacer region between RBD1 and RBD2domains. The 20-amino-acid peptide sequence GE-HARRAMQKAGRLGSTVFV corresponds to the re-gion surrounding the gap (Fig. 1).

3. The third antigenic peptide was designed for the an-tibody to recognize both splicing variants (CEARLS).It corresponds to the 20-amino-acid sequence GVER-MGPAIERMGLSMDRMV located at the C-terminusof CEAR. By analysis of the protein structure, thispeptide should be located on the outside of the celland presumably the domain incorporating this peptidecontains the CEA binding motif.

Fig. 1. The domain structure and localization of antigenic peptides on hnRNP M4/CEAR protein. The CEAR protein structure is composed of threeRNA-binding domains and a methionine-arginine-glycine-rich C-terminus; these are shown as boxes. The numbers correspond to the amino acids. Regionscorresponding to the three antigenic peptides are marked with arrows.

230 O. Bajenova et al. / Experimental Cell Research 291 (2003) 228–241

Antibodies have been produced by Research Genetics,Inc. Anti-CEAR antibodies are available from Upstate Bio-technology as “anti-CEAR receptor” antibodies. The pep-tides were attached to a KLH-carrier molecule and two NewZealand white rabbits were immunized with each antigen.The KLH-peptide was emulsified by mixing with an equalvolume of Freund’s adjuvant and injected into three subcu-taneous dorsal sites, for a total of 0.1 mg peptide perimmunization. Animals were bled from the auricular arteryaccording to the immunization schedule at 0,2,4,6,8,10, and12 weeks. The blood was allowed to clot and the serum wascollected by centrifugation and stored at �20°C.

Partial purification of anti-CEAR antibody: ammoniumsulfate fractionation

Cold (4°C) saturated ammonium sulfate in water wasadded dropwise to the antibody solution (serum) to achievea concentration of 45% saturation (v/v). The precipitatedimmunoglobulins were allowed to stand at 4°C for 2 h andthe precipitate was collected by centrifugation. The precip-itate was washed twice with 50% saturated ammoniumsulfate. The pellet was dissolved in the minimum amount ofPBS and dialyzed overnight (4°C) against 500� its volumeof PBS. A second dialysis was done the next day. Thismaterial was used for Western blotting and FACS analysis.

FACS analysis

Cells were collected, washed with PBS, and resuspendedin a concentration of 1� 106/ml FACS buffer (Hanksbuffer, 3% FCS and �NaN3). The supernatant was removedby 10-min centrifugation at 1500 rpm and replaced with 0.5ml of FACS buffer containing 10–25 �l of human IgG toblock Fc receptors. The cells were incubated for 10–15 minat 4°C. A total of 50 �l of 1:1000 anti-CEAR primaryantibody was added per sample. Cells were incubated anadditional 30 min at 4°C following three washes with FACSbuffer. After the final wash, the cell pellet was resuspendedin 100 �l of secondary anti-rabbit FITC labeled antibody in1:1000 dilution for 30 min at 4°C, stained, washed, andanalyzed.

Prokaryotic expression CEAR in Escherichia coli

The full-length human CEAR in pGEM plasmid was agift from Dr. M. Swanson. The CEARS form was isolated asthe CEA receptor and cloned into the EcoRI/XhoI sites ofthe pBK-CMV vector (Stratagene) [18]. To express theprotein the cDNA’s were subcloned in pGEX4T-3 vectorand introduced into BL21 E. coli host bacteria strain. Thetransformed cells were cultured in LB medium to a densityOD600 � 0.5 after which IPTG (0.3 mM) was added and thecells were maintained at 37°C for 2 h. Cells were homog-enized in RIPA buffer and protein lysates were subjected to

PAGE electrophoresis on 10% gel followed by Westernblotting into PVDF membranes.

Western blotting

Rat Kupffer cells and macrophage cell lines (1 � 107)were washed in PBS and then lysed in RIPA buffer (SantaCruz) with protease inhibitors (1 mM sodium orthovana-date, 2 mM sodium fluoride, 10 �g/ml leupeptin, and 0.5mM PMSF). Bacterial cells were lysed by sonication withprotease inhibitors. Cell lysates were centrifuged at 15,000rpm for 10 min at 4°C. Supernatants were collected andprotein concentrations were measured using the BCAmethod (Pierce). Equal amounts of protein were loaded onSDS–PAGE. After electrophoresis, samples were trans-ferred onto Sequi-Blot-PVDF membranes (Bio-Rad Labo-ratories). The membranes were incubated with the primaryanti-CEAR antibody in TBS buffer (1:500 dilution) washed,and incubated with a secondary anti-rabbit horseradish per-oxidase antibody complex (1:2000 dilution) (Sigma). De-tection of the protein was carried out using the ECL tech-nique (Amersham Life Science, Inc.).

Immunofluorescent analysis

Cells grown on glass coverslips were washed in PBS andfixed in 4% paraformaldehyde (in PBS) for 10 min at roomtemperature. After two washes with PBS, cells were perme-abilized with 0.25% Triton X-100/PBS/1% bovine serumalbumin (BSA) for 15 min. They were washed three timeswith PBS and incubated overnight at �4°C with primaryantibodies in PBS/1% BSA in a humid chamber. Afterseveral washes with PBS, incubation with the appropriatesecondary antibody was performed for 1 h at room temper-ature. Coverslips were washed three times in PBS andmounted with gelvatol. Antibodies were as follows: hnRNPM4 anti-rabbit (1:200) followed by goat anti-rabbit fluores-cein conjugated antibody (1:2000, Jackson Lab). In all im-munohistochemistry and FACS assays, controls includingpreincubation with preimmune serum resulted in no signal.Images were obtained using a Leitz Dialux 22 fluorescencemicroscope and processed by Adobe Photoshop software.

RT-PCR

One-step RT-PCR was performed using the SuperscriptTM One -Step RT-PCR with Platinum Tag system (Invitro-gen) to detect CEAR mRNA. Briefly, a 50-�l reaction wasprepared containing 500 ng of template mRNA, and finalconcentrations of 200 mM of each dATP, dCTP, dGTP, anddTTP, 5 mM dithiothreitol, 1.5 mM MgCl2, 400 nM of eacholigonucleotide primer (forward, 5�-GGAAGGCCACT-GAAAGTCAA-3�; reverse, 5�-TCCACGACTTTTCCCAT-CTT-3�), 1� RT-PCR buffer, and 1 �l of enzyme mixturecontaining avian myeloblastosis reverse transcriptase, Taq,and Pwo DNA polymerases. The following PCR cycling


was used: 1 cycle of 50°C 30 min, 1 cycle 94°C for 2 min;40 cycles of 94°C for 15 s, 55°C for 30 s, 72°C for 1 min;and a final extension cycle of 72°C for 10 min. The primerswere designed surrounding the deletion region encodingamino acids 187 and 226 of the rat hnRNP M4 protein. Thisgenerated two expected PCR products of 321 and 204 bprepresenting the full-length hnRNP M4 and hnRNP M4with the deletion mutation [18]. As a control for genomicDNA contamination, PCR reactions that included eachcDNA synthesis reagent except reverse transcriptase wereset up in parallel. At least two independent experimentswere performed for each amplification.

Results

CEAR is localized to the surface of Kupffer cells

To determine the CEAR cellular localization we devel-oped isoform-specific anti-CEAR antibodies as described

under Experimental procedures section. Cells were isolated,fixed, and stained on glass slides with the antibodies (Fig.2). Immunofluorescent analysis revealed that both CEARprotein isoforms are localized on the surface of rat KC (Fig.2A and B). Cells exposed only to the secondary antibodyremained negative (not shown). Treatment with 5 �g/mlCEA for 1 h enhanced CEAR fluorescence and clustering onthe surface of KC (Fig. 2C and D). The level of CEARprotein surface expression was measured by FACS analysis.KC were isolated, harvested, and vitally stained with theantibodies specific to the CEARS, CEARL, and both iso-forms. Fig. 3 (B, C, and D) shows that up to 76% of the cellswere positive in staining. When cells were exposed only tothe secondary FITC antibody (Fig. 3A–D, line 2) or with thenonspecific CD138 antibody (Fig. 3A, line 3) up to 99.6%of the cells were negative. The data indicate that both CEARisoforms are expressed on the surface Kupffer cells andsupport emerging evidence that hnRNPs, in addition to theirtraditional role in RNA biogenesis, can be involved in cellsignaling.

Fig. 2. CEAR is localized on the surface in Kupffer cells. Rat Kupffer cells were activated by CEA and stained with the isoform-specific anti-CEARN-terminal antibodies. Confocal microscopy data show that both CEAR isoforms are expressed on the surface in Kupffer Cells (A, B) with the increase inintensity and clustering upon CEA stimulation (C, D). As a control, preimmunization serum was used and was negative (not shown).


Intracellular distribution of CEAR protein inmacrophages and other types of cells

Previously a nuclear localization of hnRNP M4 protein(CEAR) was reported in Hela cells [26–31] and a variety ofembryonic mouse tissues [33]. We examined CEAR expres-sion in a number of cell types using anti-CEAR isoform-specific antibodies. The results demonstrate (Fig. 4A) ahomogeneous, diffuse distribution of CEARS proteinthroughout the nucleus and the cytoplasm in Hela, MIP101,and MIP101 (clone 17) cancer cells and in BHK&92 kidneycells. However, in p388D1 macrophages the CEARS formhas a nuclear localization with some perinuclear staining.

The CEARL protein (Fig. 4B) has a nuclear localization inHela and MIP 101 colon cancer cells and is distributedthroughout the nucleus and the cytoplasm in p388D1 macro-phages and in BHK92 kidney cells. In MIP 101 (clone 17), theCEA-producing colorectal carcinoma cell line, the cytoplasmiclevel of CEARL is intensified in comparison with the parentalnon-CEA-producing MIP101 cells. These data illustrate sur-face, nuclear, and cytoplasmic tissue-specific localization ofCEAR splicing variants and indicate that distinct functionsmay be assigned for these proteins in different cell types.

CEAR expression in E. coli

To test the specificity of anti-CEAR antibodies the cDNAsfor CEARL and CEARS isoforms were expressed in E.coli inthe GST system. Fig. 5 represents the consequent steps ofCEAR purification. The BL21 E.coli cells were transfectedwith the control pGEX4T-3 vector and a vector containingCEARS cDNA. The crude lysate of the samples, washed GSTbeads, and eluted purified protein was resolved on the gel. TheWestern blotting data show that the anti-CEARSL (Fig. 5A)and anti-CEARS (Fig. 5B) antibodies are specific to theCEARS isoform and do not recognize bacterial proteins. Theanti-CEARL antibody was not cross-reactive to the short iso-form and reacted only with CEARL. The anti-CEAS antibodydid not cross-react with the CEARL isoform (not shown).

Expression of full-length CEAR isoform induces CEAbinding in peritoneal macrophages

To elucidate the role of CEAR splicing variants inCEA uptake we looked specifically at CEAR mRNAfrom isolated rat Kupffer cells and resident peritonealmacrophages. Previously it was determined that, in con-

Fig. 3. Specificity of new anti-CEAR antibodies to Kupffer cells shown by FACS analyses. Kupffer cells were stained with anti-CEAR antibodies (B–D; line5 against short form; line 6, long form; and line 7, both forms). As a secondary antibody we used anti-rabbit–FITC conjugated antibody (A–D; line 2). Whencells were stained only with the secondary FITC antibody or with the nonspecific CD138 antibody (A), more than 99.6% of Kupffer cells were negative. Withthe anti-CEAR antibodies up to 76% of the cells had positive surface staining (B–D).


trast to Kupffer cells, resting peritoneal macrophages donot bind CEA, unless they are stimulated [34]. UsingRT-PCR primers to amplify the RBD1–RBD2 deletionregion it was determined (Fig. 6, lanes 2 and 4) that ratKupffer cells express both CEAR mRNA forms whileresident peritoneal cells express only CEARS mRNA.Stimulation of the peritoneal cells with 1 �g/ml endo-toxin (LPS) resulted in expression of the long form ofCEAR (Fig. 6, lanes 2 and 4) and these cells could nowbind CEA. These data would imply that the surfacereceptor is the full-length CEAR. Conversely, Kupffercells treated with LPS lost their ability to express the longform of CEAR and a decreased capacity to bind CEA(Fig. 6, lane 3). In summary, the full-length hnRNP M4splicing variant can play a role as a CEA receptor inperitoneal macrophages and its mRNA expression can betranscriptionally regulated in response to LPS.

Transfection CEAR cDNA in macrophage cell lines caninduce CEA binding

Previously we have shown that none of the macrophagecell lines is able to take up labeled CEA in vitro andtransfection of CEARS cDNA can induce CEA binding inp388D1 macrophages [18]. To compare both isoforms wecarried out in vitro binding experiments with 125I-labeledCEA. Five cell lines (Fig. 7), CRL2192, P388D suspensionand adherent cells, IC21, and Raw 246 cells, were tested.The cells were incubated with 125I-labeled CEA (5 �g/ml)for 15, 30, 45, and 60 min. CEA uptake by isolated ratKupffer cells was used as a positive control. After 48 h, 125ICEA binding was observed in transfected PD388D1 cells(Fig. 7). Transfectants containing matching concentrationsof the empty pGEX4T-3 vector (not shown) did not bindCEA. Transfection of CEARL cDNA into P338D1 cells

Fig. 4. Differences in CEAR cellular localization in macrophages and cancer cells. Different cultured cell types were stained with the anti-CEARL andanti-CEARS antibodies. A. The results show that in Hela, MIP101, and BHK92 kidney cells the CEARS form has a nuclear–cytoplasmic localization. Incontrast, in p388D1 cells CEARS has a nuclear and in Kupffer cells a surface localization. B. The CEARL isoform is localized in the nuclei in Hela andMIP101 cells and on the surface in Kupffer cells. It has nuclear–cytoplasmic patterns in p388D1 and BHK92 cells and MIP101 (clone 17), a stable cell linetransfected with the full-length cDNA for CEA. The CEARL protein level in the CEA-producing MIP101 (clone 17) cells shows a higher expression thanin the parental MIP101 line.


showed greater CEA binding than transfectants producedwith the CEARS cDNA (Fig. 7). We propose that the myri-stylation signal that is present in the full-length protein andabsent from the deleted form may effect CEAR intracellularlocalization and influence CEA binding as its role in proteintargeting to the plasma membrane [32]. Using an adherentform of P388D we were able to increase the efficiency oftransfection such that CEA binding approached that ofKupffer cells. These results demonstrate that the cDNAtransfection of both CEAR isoforms is effective and suffi-cient to induce CEA binding in p388D1 macrophages andthe full-length form is more potent in binding CEA.

Other macrophage cell lines transfected under the sameconditions did not initiate CEA binding. One of the possiblereasons for the lack of response may be the difference inCEAR expression. Using RT-PCR primers to amplify theRBD1–RBD2 truncated region it was determined (Fig. 8A)that, similar to Kupffer cells, two mRNA PCR products areexpressed in most macrophage cell lines with the variableratio between CEARS and CEARL. Alternatively, threesplicing isoforms were detected in pH1 and U937 cells.These cells have an extra PCR product with a size greaterthan 321 bp (Fig. 8A). In Kupffer cells the CEARS isoformis the most abundant and this may explain why the CEARS

form was isolated as a CEA receptor. Western blot analysisrevealed (Fig. 8B) two forms of CEAR protein (p76, p80)with the variable ratio between them. Both CEAR isoformsare overexpressed in p388D1 in comparison with othermacrophage cell lines. These results show significant dif-

ferences in CEAR mRNA and protein expression betweenmacrophages; this can influence CEA binding.

Effect of transfection on CEAR protein expression andlocalization in p388D1 cells

We hypothesized that transfection induces surface ex-pression of CEAR protein that initiates CEA uptake. To testthis hypothesis adherent p388D1 cells were transfected withthe CEARS cDNA (Fig. 9A, lines 1 and 2) or an emptypGEX4T-3 vector (Fig. 9A, line 3, negative control). Cellswere harvested and vitally stained with the C-terminal anti-CEAR antibody specific to both isoforms (anti-CEARSL)(Fig. 9A, line 2). These results demonstrate increased sur-face expression of the CEAR protein as a result of trans-fection (Fig. 9A, line 2). The empty pGEX4T-3 vector didnot have any effect (Fig. 9A, line 3).

The observation of individual cells by confocal micros-copy data (Fig. 9B) revealed that the transfection effectsCEAR isoforms expression and localization. Introduction oflong-form CEAR cDNA results in cytoplasmic localizationof both forms (Fig. 9B, 5 and 6). Transfection with theshort-form CEAR cDNA had no effect on the expression ofthe short form (Fig. 10B) and seems to trigger nuclearlocalization of the full-length protein (Fig. 10). To summa-rize, overexpression of one splicing form can effect bothlocalization and expression of another form. These datademonstrate a common regulatory mechanism between

Fig. 6. Changes in CEAR isoforms mRNA expression induce CEA bindingin peritoneal macrophages. CEAR mRNA expression in rat Kupffer cellsand peritoneal macrophages have been tested by RT-PCR. The primerswere designed surrounding the RBD1–RBD2 deletion region encodingamino acids 187 and 226 of the rat CEAR protein. This generated twoexpected PCR products of 321 and 204 bp representing CEARL andCEARS. Resting peritoneal macrophages do not respond to CEA unlessthey are stimulated [34]. In control they express only the CEARS isoformmRNA (line 4). Stimulation with LPS (1 �g/ml) for 90 min results in theexpression of the CEARL mRNA (line 6). These cells are now capable ofCEA binding. Rat Kupffer cells express both forms while LPS treatmentreduces expression of the CEARL mRNA (line 3).

Fig. 5. The consequent steps in CEAR protein purification. The CEARcDNA was expressed in E. coli host strain in the pGEX4T-3 GST vector.The BL21 E. coli cells were transfected with the control pGEX4T-3 vectorand a vector containing CEARS cDNA. The crude lysate of the samples,washed GST beads, and eluted purified protein were resolved on the gel.Cells were lysed and a protein expression was tested in a Western blot withthe C-terminal anti-CEARSL (A) and N-terminal anti-CEARS (B) antibod-ies. The Western blotting data show that the anti-CEARSL (A) and anti-CEARS antibodies are specific to CEARS isoform and do not recognizebacterial proteins. The anti-CEARL antibody was not cross-reactive to theshort isoform and reacted only with CEARL (not shown). The anti-CEAS

antibody did not cross-react with the CEARL isoform.


CEAR splicing forms and that there is a balance betweenCEAR isoforms that is important for CEA binding.

The effect of CEA on CEAR protein expression andlocalization

To elucidate the effect of CEA treatment on Kupffercells, transfected p388D1 macrophages cells were stainedwith the anti-CEAR antibodies. Fig. 2 shows that the expo-sure of KC to 5 �g/ml CEA for 1 h induces both CEARsurface expression and clustering (Fig. 2C and D).

In transfected p388D1 cells CEA treatment induces cy-toplasmic localization of CEARS versus nuclear localizationin control (Fig. 10C). The CEARL isoform in control cellsis diffusely distributed through the nucleus and the cyto-plasm (Fig. 10B). CEA exposure significantly enhances thelevel of CEAR expression (Fig. 10D). These results dem-onstrate that CEA exposure induces overexpression andrelocation of CEAR isoforms and imply that the CEARS

isoform can shuttle between the nucleus and the cytoplasm.

Discussion

This study supports emerging evidence that hnRNPs, inaddition to their traditional role in RNA biogenesis, can beinvolved in cell signaling [22–25,30,31]. HnRNP M4 it isnot the first nuclear protein of this type to be identified as acell surface receptor. Several reports show that hnRNP Uand nucleolin act as cell surface receptors, which, by shut-tling between the cytoplasm and nucleus, may provide amechanism for extracellular regulation of cell growth orvirus infection [35,36]. In contrast to the current opinionabout the nuclear localization and function of hnRNP Mproteins [26,27,29,30,33], this study provides evidence forsurface localization of the hnRNP M4 protein in Kupffercells. To elucidate the functional differences betweenCEAR splicing variants we developed isoform-specific an-tibodies. The data show that both CEAR isoforms are ex-pressed on the Kupffer cell surface. This surface localiza-tion allows binding of CEA, activation of Kupffer cells, andan increased potential for CEA-producing colorectal cancer

Fig. 7. Comparative CEA uptake of KC and macrophage cell lines. Nonresponsive to CEA macrophage cell lines: CRL2192, P388D suspension and adherentcells, IC21, and Raw 246 cells were transfected with the cDNAs encoding the CEARL and CEARS. The CEA uptake was measured as the amount of iodinecounts per cell. After 48 h cells were incubated with the iodine labeled CEA (5 �g/ml) for 15, 30, 45, and 60 mins. Analysis revealed that transient transfectionof CEAR cDNA into p388D1 macrophages results in CEA binding. Both constructs were efficient. The highest level of CEA uptake is seen with KC andCEARL. An approximately 2 times increase in CEA uptake was observed with adherent (ad) cells versus suspension (s) p388D1 cells. The adherent cellshave up to 70% and the suspension cells up to 30% of the capacity of rat Kupffer cell CEA uptake.


cells to initiate hepatic metastases. The full-length humanhnRNP M4 has been described previously as a receptor forthyroglobulin [37,38]. In this case the binding site is likelyto be different from the CEA site, illustrating the multifunc-tional nature of this protein.

Initially, the truncated form of hnRNP M4 (CEARs) wasisolated as the CEA receptor. However, cDNA transfectionexperiments in nonresponsive macrophage cell lines re-vealed that both CEAR isoforms induce CEA binding.Moreover, several observations suggest that the full-lengthprotein is more potent as a CEA receptor. (a) Transfectionof CEARL cDNA induces a higher level of CEA bindingthan CEARS in vitro in p388D1 cells. (b) CEARL has asurface and a cytoplasmic localization in p388D1 cells ver-sus nuclear localization of CEARS. (c) CEA binding inactivated peritoneal macrophages correlates with the induc-tion of the CEARL protein isoform.

To identify structural protein motif/s the RBD1–RBD2region of 39 amino acids that is different between twoisoforms was analyzed using the Prosite data base. Werecognized a unique motif that is a myristoylation signalPGGPGMITIP. Myristoylation, a covalent attachment offatty acids to proteins, is now a widely recognized form ofprotein modification. Fatty acetylated proteins play a keyrole in regulating cellular structure and function [32]. It wasshown that the presence of myristate at the amino terminushelps target the protein to membranes and is essential for thefunction of a number of kinases [39], including Src [40],c-Abl [41], and cGMP-dependent protein kinase [42]. Theaccessibility of the myristic group for membrane binding is

regulated in some proteins by a switch mechanism that canbe triggered by phosphorylation, calcium binding, cleavageof the myristoylated protein, or GTP binding [43]. Forexample, myristoylated Hsp90N protein associates with Rafand targets it to the plasma membrane, thus substituting forc-Ras in this role. Thus, the chaperone family, which wereheretofore thought to be involved mostly in protein folding,can function as oncogenes [44]. We suggest that myristoy-lated CEAR may target to a plasma membrane for CEAbinding and Kupffer cell activation. This hypothesis needsfurther investigation to elucidate whether the target recog-nition is impaired by mutations altering the myristoylationmotif.

Furthermore, earlier work of ours (E.S. Fox and P.Thomas, unpublished data) using 13C NMR spectroscopyrevealed that Kupffer cell plasma membranes were morefluid than those of the peritoneal macrophage. Such changesin the lipid bilayer could allow for the insertion of proteinsinto one cell type and not the other. Alternatively, Kupffercells may express associated proteins that are not present inthe other macrophages that allow CEAR insertion intoplasma membranes.

This report demonstrates that in response to CEA thedeleted form of hnRNP M4 protein in p388D1 cells canshuttle from the cytoplasm to the nucleus. This shuttlingability resembles that of hnRNP A1, hnRNP B, and hnRNPK proteins. For hnRNP A1 it was reported that exposure ofdifferent cell lines (3T3, COS, 293) to stress stimuli (os-motic shock, UV irradiation), but not to mitogenic activa-tors such as PDGF or EGF results in a marked cytoplasmic

Fig. 8. CEAR mRNA and protein expression in KC and other macrophages. A. CEAR mRNA expression in Kupffer cells and six macrophage cell lines: PH1,U937, CRL2192, Raw 246.7, IC21, and adherent P388D1 have been tested by RT-PCR. The primers were designed surrounding the deletion region encodingamino acids 187 and 226 of the rat CEAR protein. This generated two expected PCR products of 321 and 204 bp representing the full-length CEAR and thatwith the deletion mutation. The data show that in human and rat Kupffer cells two isoforms are present. CEARS is the most abundant in Kupffer cells. Upto three splicing variants are characteristic for PH1 and U937 cells. In P388D1, CRL2192, Raw 246.7, and IC21 macrophages two CEAR mRNA types arepresent and the ratio between them varies considerably.


accumulation of hnRNP A1 concomitant with an increase inits phosphorylation [45]. In support of this observation, ourstudy provides evidence that CEA can influence posttran-scriptional events, including CEAR alternative splicing.This regulatory mechanism may turn out to be a generalone, by which different signaling pathways may effect thenuclear ratio of specific antagonistic splicing regulators,thereby modulating in this case the transcription of IL1-�,IL6, IL10, and TNF-� cytokines.

The regulation and functions of hnRNP M4/CEAR splic-ing as well as many functions of hnRNP M proteins are farfrom being understood. Recent progress in uncovering themolecular strategies that control alternative splicing has ledto the identification of many factors that influence the se-lection of the alternative splice sites [46]. Individual mem-bers of the hnRNP and serine-arginine (SR) families ofproteins have antagonistic effects in regulating alternativesplicing [47]. Our data support the observation that thebalance between these key elements of splicing machinerycontrols splice site selection and has profound implicationson the production of protein isoforms with different func-tions. Taken together they indicate that the balance between

CEARL and CEARS and their intracellular localization arekey elements for CEA binding. CEA exposure inducesCEAR up-regulation in Kupffer cells and modulates alter-native splicing by altering the ratio and localization ofCEAR mRNAs. These data are in support of the growingevidence that splicing decisions are regulated by a variety ofpathways such as those activated by cell surface receptors,stress, and other mechanisms [45].

CEA is a widely used tumor marker but its role in normalhuman physiology versus cancer remains unknown. Func-tions for CEA have been suggested in the areas of intercel-lular cell adhesion [48], signal transduction [7], host–mi-crobial interaction [49], metastasis [7], and apoptosis [50].This variety of functions creates a multiplicity of peptideand carbohydrate epitopes that can be potential nonspecifictargets for anti-CEA immunotherapy and can explain whyanti-CEAR antibodies used in clinical trials have not shownany definitive benefits against advanced cancers. BecauseCEA binding to and activation of Kupffer cells allowscolorectal cancer to implant and survive in the liver, a betterunderstanding of the mechanisms of CEAR expression andfunction will open a window of opportunity to design spe-

Fig. 9. Effect transfection on CEAR mRNA expression and cellular localization in p388D1 macrophages. A. Increase in CEAR surface expression intransfected p388D1 macrophages showed by FACS analyses. p388D1 cells transiently transfected with CEARS were vitally stained with the anti-CEARC-terminal antibody. The data show that transfection increases the amount of cells in the population specific to the anti-CEAR antibody (lane 2). It can occuronly if receptor is expressed on the surface of the cells. Nontransfected cells (lane 1) and cells stained with the secondary antibody (lane 3) remained negative.B. Control and transfected P388D1 cells were stained with the anti-CEAR isoform-specific antibodies. In control the CEARS isoform is detected in the nuclei(B-1). Transfection with CEARS cDNA enhances the level of CEARS nuclear staining (B-3). Transfection with CEARL cDNA results in cytoplasmicexpression CEARS isoform around the nuclei (9B-3). Staining with anti-CEARL antibody revealed changes in the localization of full-length isoform intransfected cells (B-4,6) versus control (9B-2).


cific anti-CEAR therapies against the metastatic spread ofCEA overexpressing cancers.

Acknowledgments

This work was supported by National Institutes of HealthGrant CA 74941 (to P.T.). The costs of publication of thisarticle were defrayed in part by the payment of pagecharges. The article must therefore be hereby marked “ad-vertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact. The C-terminal anti-CEAR an-tibody is commercially available from Upstate Biotechnol-ogy, Product No. 07-296 (anti-CEA receptor). We are grate-ful to Dr. Hartmut Juhl for critical reading of the manuscript

and helpful discussion and to Dr. M. Swanson for thefull-length human hnRNP M4 cDNA vector.

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