©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Heregulin Activation of Extracellular Acidification in Mammary Carcinoma Cells Is Associated with Expression of HER2 and HER3 (*)

(Received for publication, June 26, 1995)

Samuel D. H. Chan (1) Diana M. Antoniucci (1)(§) Katherine S. Fok (1) M. Liisa Alajoki (1) Richard N. Harkins (2) Stuart A. Thompson (2) H. Garrett Wada (1)(¶)

From the  (1)Molecular Devices Corporation, Sunnyvale, California 94089 and (2)Berlex Biosciences, Richmond, California 94804

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

HER2, the erbB-2/neu proto-oncogene product, is a 185-kDa transmembrane glycoprotein related to the epidermal growth factor receptor. Overexpression of HER2 was reported in several human adenocarcinomas, including mammary and ovarian carcinomas. A family of glycoproteins, the heregulin/neu differentiation factors, was characterized and implicated as the ligands for HER2. Recently, it has been shown that HER2 alone is not sufficient to reconstitute high affinity heregulin receptors and that HER3 or HER4 may be the required components of the heregulin receptors on mammary carcinoma cells (Sliwkowski, M. X., Schaefer, G., Akita, R. W., Lofgren, J. A., Fitzpatrick, V. D., Nuijens, A., Fendly, B. M., Cerione, R. A., Vandlen, R. L., and Carraway, K. L., III(1994) J. Biol. Chem. 269, 14661-14665; Plowman, G. D., Green, J. M., Culouscou, J.-M., Carlton, G. W., Rothwell, V. M., and Buckley, W.(1993) Nature 366, 473-475). Using the Cytosensor to measure the extracellular acidification rate, we have examined the effects of recombinant human heregulin-alpha on three mammary carcinoma cell lines expressing HER2 (MDA-MB-453, SK-BR-3, and MCF-7), an ovarian carcinoma cell line expressing HER2 (SK-OV-3), and CHO-K1 and 293-EBNA cells stably transfected with HER2. By reverse transcription polymerase chain reaction and Western blotting, we found that the breast cells also express HER3 and that the ovarian line co-expresses the HER4 message. A dramatic increase in the acidification rate was observed for the mammary carcinoma cells co-expressing high levels of HER2 and HER3. In contrast, the ovarian cells expressing high levels of HER2 and low levels of HER4 or CHO-K1 and 293-EBNA cells expressing HER2 alone were not responsive to heregulin. When these same transfected cells were exposed to monoclonal anti-HER2 antibody followed by anti-IgG to cause aggregation of the HER2 molecules, an increase in the acidification rate was observed, indicating coupling of transfected HER2 to the signal transduction pathway. Transfection of HER2 into MCF-7 cells, on the other hand, gave 4-fold enhanced acidification responses. These data, together with the previously reported high affinity heregulin binding and activation of tyrosine phosphorylation in HER2 and HER3 co-transfected cells support the role of HER2 and HER3 as components of the heregulin receptor in breast cells.


INTRODUCTION

A family of EGF (^1)receptor-related proteins has recently been described that includes the proto-oncogene products of erbB-2/neu (HER2), erbB-3 (HER3), and erbB-4 (HER4)(1, 2) , which have a high degree of sequence homology (40-50%) and similar molecular structure, containing a glycosylated extracellular domain, a transmembrane domain, and a conserved tyrosine kinase domain. The HER2 receptor was the first of these discovered by two groups who screened human genomic libraries at low stringency using EGF receptor-based probes(3, 4) . Subsequent to this, the HER3 (5) and HER4 (6) receptors were identified using similar techniques. Despite the structural similarity between these receptors, they have no binding affinity for any of the EGF ligands, which leaves open the possibility that ligands exist for each of these receptors.

The identity of the ligands for these receptors has been the object of much investigation resulting in the identification of several candidate ligands for HER2. A 30-kDa protein (7) and heregulin (8) were purified from the conditioned medium of MDA-MB-231 mammary carcinoma cells. Neu differentiating factor was purified from the conditioned medium of ras-transformed fibroblasts(9) , and Neu activating factor was purified from the conditioned medium of human ATL-2 T cells(10) . Each of these putative ligands was shown to activate phosphorylation of the HER2 protein, p185. Subsequent to the cloning of Neu differentiating factor (11) and heregulin(8) , peptide ligands with a high degree of sequence homology with heregulin/Neu differentiating factor, the glial growth factors-I, -II, and -III (12) and a protein that stimulates muscle acetylcholine receptor synthesis (ARIA) (13) were identified. These findings indicate that a family of heregulin-like ligands exist that are important in the development and regeneration of the nervous system and are products of the same gene, produced by alternatively spliced mRNA(12, 13) .

Whether the heregulin/Neu differentiating factor is truly a ligand for HER2 was recently disputed by the finding that heregulin did not activate phosphorylation of p185 in ovarian cell lines expressing HER2 and in ovarian cells transfected with HER2(14) . These results suggest that another component in addition to HER2 is required for the heregulin receptor. More recently, both HER4 (16) and HER3(17, 18) have been separately reported to be the additional component required in combination with HER2 to reconstitute heregulin high affinity binding and heregulin-mediated p185 phosphorylation. We have recently found using a sensor-based microphysiometer, Cytosensor(19, 20) , that heregulin-alpha stimulates an increase in the extracellular acidification rate in SK-BR-3 cells, which express high levels of HER2(21) . Using this technique to monitor cellular metabolism, we have measured the cellular response of a series of carcinoma cell lines expressing HER2, HER3, and HER4 and cells transfected with HER2 for their metabolic response to heregulin-alpha. Our results indicate that cells expressing both HER2 and HER3 respond to heregulin, and those expressing HER2 and low levels of HER4 or HER2 alone do not.


MATERIALS AND METHODS

Cell Culture

CHO-K1 (ATCC) cells were cultured in Ham's F-12 medium containing 10% fetal bovine serum, 2 mML-glutamine, and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin). 293 (ATCC), 293-EBNA (Invitrogen), MDA-MB-453 (ATCC), MCF-7 (ATCC), SK-BR-3 (ATCC), and SK-OV-3 (ATCC) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mML-glutamine, and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin).

Construction of pCEP/HER2 Expression Plasmid and Establishment of HER2-expressing Cell Lines

A chimeric cDNA fragment containing the complete open reading frame of the human HER2 gene was constructed by splicing a 1.0-kilobase pair EcoRI-AflIII fragment of the plasmid pHER2-436-1 (59296, ATCC) and a 3.0-kilobase pair AflIII-StuI fragment of the plasmid pCER204 (57584, ATCC). The construct was subsequently cloned into pCEP4 vector (Invitrogen) downstream of the cytomegalovirus promoter to generate the pCEP/HER2 expression plasmid. The pCEP4 vector contains the hygromycin-resistant gene that allows selection of stable transformants with the antibiotic hygromycin B (CalBiochem). pCEP/HER2 was transfected into CHO-K1, 293-EBNA (Invitrogen), or MCF-7 by electroporation using a BTX electroporator according to the manufacturer's instructions. After 24 h, the cells were washed and subcultured. Hygromycin-resistant cells were cloned after exposing the transfected cells to hygromycin B (400 µg/ml) for 14 days. Clonal cell lines were screened by immunohistochemical staining using anti-neu (HER2) monoclonal antibody (Santa Cruz Biotechnology) and fluorescein isothiocyanate-labeled anti-mouse IgG. The clones with the highest HER2 expression were selected for further study.

Northern Blot Analysis

Total RNA was isolated by the guanidinium thiocyanate/phenol extraction method(24) . 20 µg of RNA were size-fractionated on 1% agarose-formaldehyde gels and blotted to nylon membranes (LabLogix). The membranes were prehybridized for 30 min at 65 °C in Universal Hybridization Solution (DNA/RNA hybridization kit, LabLogix). A 4.0-kilobase pair HER2 cDNA was labeled with biotin by random priming (BioPrime DNA labeling system, Life Technologies, Inc.), and hybridization was carried out for 18 h at 65 °C. Blots were washed at a final stringency of 0.1 times SSC and 0.1% SDS at 65 °C. Hybridization signals were detected using the Luminescent DNA/RNA detection kit (LabLogix) according to the manufacturer's instructions. In brief, the blots were incubated with streptavidin-alkaline phosphatase conjugate followed by detection with a chemiluminescent dioxetane substrate and x-ray film.

PCR Primers and Biotinylated Oligonucleotide Probes

Oligonucleotides were designed using the GeneWorks program (IntelliGenetics) and synthesized by Operon Technologies. The primer pairs amplify a 3`-fragment of the coding sequence of HER2, HER3, or HER4 using the polymerase chain reaction, and the biotinylated oligonucleotide probes specifically hybridize to internal sequences in the corresponding PCR products. For HER2, the upstream primer is TTGGACAGCACCTTCTACCG, the downstream primer is TCCTTAGGACAGGTTCCTGG, and the PCR product is 910 base pairs. For HER3, the upstream primer is TGTCAATGTGTAGAAGCCGG, the downstream primer is GCAGGAGTTACGTTCTCTGG, the PCR product is 702 base pairs, and the biotinylated probe is biotin-GGTGATTATGCAGCCATGG. For HER4, the upstream primer is CCAGAGCAAGAATTGACTCG, the downstream primer is GCTTACACCACAGTATTCCGG, the PCR product is 820 base pairs, and the biotinylated probe is biotin-ATTCTCTGCCACAATAGGCC.

RT-PCR and Southern Blot Analysis

Total RNA was isolated by the guanidinium thiocyanate/phenol extraction method and treated with DNase I (Amplification grade, Life Technologies, Inc.) to eliminate residual genomic DNA. First strand cDNA was synthesized using the SUPERSCRIPT preamplification system (Life Technologies, Inc.). PCR was carried out in a 50-µl reaction with an initial temperature cycle of 5 min at 94 °C, followed by 35 cycles of 1 min at 94 °C, 1 min at 55 °C, and 1 min at 72 °C and a final cycle of 7 min at 72 °C in a thermal cycler. The PCR buffer was composed of 20 mM Tris-HCl, pH 8.75, 10 mM KCl, 10 mM (NH(4))(2)SO(4), 2 mM MgSO(4), 0.1% Triton X-100, 0.1 mg/ml bovine serum albumin, 0.2 mM dNTPs, 0.25 µM of each primer, and 2.5 units Pfu DNA polymerase (Strategene). 20 µl of the PCR product was fractionated on a 1.2% agarose gel and blotted to nylon membranes (LabLogix). The blot containing HER2 PCR products was prehybridized and hybridized as in Northern blot analysis. For HER3 and HER4, blots were hybridized with the corresponding biotinylated oligonucleotide probes for 18 h at 37 °C, washed with 1 times SSC and 1% SDS at 37 °C, and processed for chemiluminescent detection.

Western Blot Analysis

Cells were analyzed for the expression of HER2 (p185), HER3 (p160), and HER4 (p180) using Western blotting with mouse monoclonal anti-NEU (HER2) SC08, rabbit anti-erbB-3 peptide (HER3) SC285, and rabbit anti-erbB-4 peptide (HER4) SC283 antibodies (Santa Cruz Biotechnology). Cells were harvested from T-75 flasks using EDTA in phosphate-buffered saline and centrifuged, and the cell pellet was resuspended and homogenized in 5 mM sodium phosphate, 2 mM magnesium chloride, and 1 mM phenylmethylsulfonyl fluoride, pH 7.4. The membrane fraction was isolated by centrifugation at 1,500 times g at 4 °C for 10 min to remove debris and nuclei and at 40,000 times g for 20 min to collect the microsomal pellet. The microsomes were solublized in SDS reducing sample buffer, and 20 µg of protein was electrophoresed per lane in an 8% SDS-polyacrylamide gel. After electrophoretic transfer to polyvinylidene difluoride membrane, the Western blot was processed according to the Luminescent Western blot kit instructions (LabLogix). The blot was blocked at 42 °C for 1 h and probed with 1 µg/ml anti-HER2 antibody or 1/500 dilutions of anti-HER3 and anti-HER4 peptide antibodies overnight at 4 °C, followed by horseradish peroxidase labeled anti-mouse or anti-rabbit IgG for 1 h at room temperature. The reactive bands were detected using chemiluminescent horseradish peroxidase substrate and x-ray film.

Cell Processing for Microphysiometry

Cells were released from culture flasks by incubation with 5 mM EDTA in phosphate-buffered saline and harvested by centrifugation. The cell pellets were resuspended in complete culture media and plated into sterile cell capsule cups (Molecular Devices Corp.) at 3 times 10^5 cells/capsule in a 12-well culture plate. Cells were incubated for 4 h to allow for cell adhesion to the capsule membrane, and then the medium was changed to serum-free media for an overnight period of serum starvation to reduce basal metabolic rate. A spacer gasket and capsule insert (Molecular Devices Corp.) was placed into each capsule cup. The assembled capsule cups were loaded into sterilized Cytosensor chambers. Low buffering, bicarbonate-free RPMI 1640 medium (Irvine Scientific) containing 1 mg/ml human serum albumin (Miles Labs) was used as running media in the Cytosensor, and the extracellular acidification rates were monitored as described earlier, collecting a rate measurement every 2 min(18, 19) . Cells were exposed to recombinant heregulin-alpha (Berlex Biosciences) or antibodies in running media for 15-min intervals, and the effects on the acidification rate were measured by the instrument. The acidification rate was normalized to 100% prior to the addition of test materials.


RESULTS

Extracellular Acidification Rate Response of Carcinoma Cells to Heregulin

Human mammary and ovarian carcinoma cells known to express HER2 were treated with heregulin-alpha in the Cytosensor to determine the effects of this putative HER2 ligand on cellular metabolism as measured by extracellular acidification. As shown in Fig. 1A, the rate of extracellular acidification for SK-BR-3 cells rapidly increased to 21% above controls in response to treatment with heregulin at 100 ng/ml (2.2 nM), reaching peak levels within 10 min. The amplitude of the response decreased with decreasing concentrations of heregulin, and the acidification rate increase was inhibited by a tyrosine kinase inhibitor, genistein (22) at 25-50 µg/ml (data not shown), indicating that the response was coupled through a protein-tyrosine kinase signaling pathway, as was found earlier for the EGF-receptor(19, 23) . A similar response was observed for MDA-MB-453 cells (Fig. 1B), with acidification rates increasing to 30% above controls within 20 min when treated with 80 ng/ml heregulin. The MCF-7 mammary carcinoma cells gave a much weaker response to heregulin at 100 ng/ml, peaking at only 6% above controls (Fig. 1C), and SK-OV-3 cells did not give a response to 100 ng/ml heregulin (Fig. 1D). Heregulin responsiveness and HER2 expression levels correlated for the mammary carcinoma cell lines; however, the ovarian cell line that expresses high levels of HER2 (see below) did not respond at all to heregulin.


Figure 1: Acidification response of human mammary and ovarian carcinoma cells to heregulin-alpha. Carcinoma cells were prepared and loaded into Cytosensor capsules as described under ``Materials and Methods.'' The cells were monitored in the Cytosensor until acidification rates stabilized, and then media containing various concentrations of heregulin-alpha were switched into the fluid stream for 15 min. A, SK-BR-3 cells were exposed to 100 (2.2 nM) (box), 10 (), 1 (bullet), or 0 (circle) ng/ml heregulin-alpha. B, MDA-MB-453 cells were exposed to 80 (1.8 nM) (box), 20 (), 5 (bullet), or 0 (circle) ng/ml heregulin-alpha. C, MCF-7 cells were exposed to 100 (bullet) or 0 (circle) ng/ml heregulin-alpha. D, SK-OV-3 cells were exposed to 100 (bullet) or 0 (circle) ng/ml heregulin-alpha.



Expression of HER2 in Carcinoma Cells

Northern blot analysis of total RNA using the HER2 cDNA probe showed that a 4.8-kilobase transcript was expressed in the human mammary carcinoma cells MDA-MB-453 and SK-BR-3 and in the ovarian cell SK-OV-3 (Fig. 2A). HER2 mRNA level was the highest in SK-BR-3, followed by SK-OV-3 and MDA-MB-453; it was undetectable in MCF-7 mammary carcinoma cells. The results from Northern blot analysis were confirmed by the Western blot (Fig. 2B) and immunofluoresence analysis (data not shown), which indicated p185 at high levels in SK-BR-3, SK-OV-3, and MDA-MB-453 cell membranes. A very faint p185 band was observed in the MCF-7 Western blot, and no detectable p185 band was observed in blots of 293 and CHO-K1 membranes.


Figure 2: Expression of HER2 and HER3 in human mammary and ovarian carcinoma cells. Human mammary and ovarian carcinoma cell lines were tested for the presence of HER2 mRNA by Northern blot analysis and p185 by Western blot analysis. A, total RNA (20 µg) isolated from human mammary carcinoma cells (MDA-MB-453, MCF-7, and SK-BR-3), human ovarian tumor cells (SK-OV-3), and human kidney cells (293) was fractionated in a 1% agarose-formaldehyde gel. After transferring to a nylon membrane, the blot was hybridized with a biotinylated HER2 cDNA probe. The hybridized blot was incubated with a streptavidin-alkaline phosphatase conjugate followed by chemiluminescent detection as described under ``Materials and Methods.'' B, the membrane fraction isolated from the same breast and ovarian cell lines were solublized and electrophoresed in SDS-polyacrylamide gels for Western blot analysis as described under ``Materials and Methods.'' The p185 bands were detected using monoclonal anti-HER2 antibody followed by horseradish peroxidase-labeled anti-IgG and chemiluminescent detection. The faint p185 band for MCF-7 is invisible in the photograph, but it is present on the original film. C, Western blot analysis of the mammary and ovarian cell lines for HER3 using anti-HER3 peptide rabbit antibody was conducted as described for B. The p160 bands corresponding to HER3 were detected by chemiluminescent detection.



RT-PCR Detection of HER2, HER3, and HER4 mRNA Expression in Carcinoma Cells

An initial attempt to detect HER3 and HER4 mRNA by Northern blot analysis using synthetic oligonucleotide probes was unsuccessful. However, using RT-PCR to amplify the mRNA sequences, we were able to demonstrate expression of much greater HER3 mRNA in the mammary carcinoma cells MDA-MB-453, MCF-7, and SK-BR-3 in comparison with ovarian carcinoma cells, SK-OV-3 (Fig. 3A). This result was confirmed by Western blot analysis using anti-HER3-specific antibody, which detected p160 in the breast cells but not in the ovarian cells (Fig. 2C). In contrast, expression of the HER4 message was very low or not detectable in the mammary carcinoma cells by RT-PCR, and SK-OV-3 was the cell line that expressed the highest level of HER4 mRNA. The inability to detect the p180 HER4 protein by Western blotting of SK-OV-3 samples indicated that HER4 levels were low in these cells (data not shown). RT-PCR confirmed that HER2 mRNA transcripts were present in both mammary and ovarian carcinoma cells (Fig. 3A) and demonstrated that MCF-7 cells also expressed the HER2 message in agreement with the Western blot analysis. We were, however, unable to detect its expression using the less sensitive Northern blot analysis. The authenticity of the PCR products was confirmed by Southern blot analysis using biotinylated HER2, HER3, or HER4 probes (Fig. 3B) and by restriction digestion analysis (data not shown).


Figure 3: RT-PCR detection of HER2, HER3, and HER4 mRNA expression in human mammary and ovarian carcinoma cells. Total RNA was isolated and RT-PCR was performed as described under ``Materials and Methods.'' A, the ethidium bromide-agarose gels of the PCR products using primers for HER2, HER3, and HER4 are shown here. The DNA size standard X174/HaeIII is also included. B, PCR products from A were blotted to a nylon membrane, probed with the corresponding biotinylated probes, and detected as described under ``Materials and Methods.''



Overexpression of HER2 in CHO-K1, 293-EBNA, and MCF-7 Cells Stably Transfected with pCEP/HER2

Hygromycin B-resistant clonal cell lines stably transfected with pCEP/HER2 and expressing high levels of HER2 gene product as observed by immunohistochemical staining using a fluorescein-labeled HER2 antibody were selected for Northern blot analysis. High levels of HER2 message expression were detected in CHO/HER2-H, 293/HER2-O, and 293/HER2-T but not in the parental cells (Fig. 4A). CHO/HER2-O, a hygromycin B-resistant clonal cell line that did not express HER2 protein as determined by immunohistochemical staining, was included as a negative control. As indicated above, we were unable to detect the expression of HER2 mRNA in MCF-7 cells by Northern blot analysis, but it was possible by RT-PCR. On the other hand, overexpression of the message was demonstrated in MCF-7 cells stably transfected with pCEP/HER2 (MCF/HER2-A, Fig. 4A) in Northern blots. Immunochemical staining with an anti-HER2 antibody (data not shown), Western blot analysis (Fig. 4B), and subsequent Cytosensor testing confirmed the increased expression of HER2 protein in the MCF/HER2-A cells. Like the parental MCF-7 cells, expression of HER2 mRNA in MCF/HER2-C, a transfected cell line that did not overexpress the protein, was below the detection limit of Northern blot analysis.


Figure 4: Overexpression of HER2 RNA in CHO-K1, 293-EBNA, and MCF-7 stably transfected with pCEP/HER2 expression plasmid. Clonal cell lines stably transfected with pCEP/HER2 plasmid was isolated as described under ``Materials and Methods.'' A, Northern blot analysis of total RNA was performed using a biotinylated HER2 cDNA probe. SK-BR-3 RNA was included as a positive control for HER2 mRNA expression. B, Western blot analysis of membrane fractions of CHO-K1, 293-EBNA, and MCF-7 cells transfected with pCEP/HER2 was performed as described under ``Materials and Methods.'' The p185 bands were detected using monoclonal anti-HER2 antibody followed by horseradish peroxidase-labeled anti-IgG and chemiluminescent detection. SK-BR-3 was included as a positive control.



Lack of Acidification Response by HER2-transfected CHO-K1 and 293 Cells to Heregulin

In order to further examine the relationship between the cellular response to heregulin and HER2 expression, CHO-K1 and 293 cells that were transfected with the pCEP/HER2 expression vector and selected for high HER2 expression were tested in Cytosensor for cellular response to heregulin-alpha treatment. CHO/HER2-H and 293/HER2-O cloned cell lines gave no significant response to 100 ng/ml heregulin (Fig. 5, A and C). The functionality of the transfected tyrosine kinase receptors was evaluated by activating them by aggregation with anti-HER2 followed by anti-mouse IgG. It has been demonstrated that antibody dimerization and aggregation of HER2 activates autophosphorylation of p185(14, 25) . As shown in Fig. 5, B and D, the aggregation of the HER2 in the CHO and 293 cells activated increases in acidification rate of approx30% for the CHO/HER2-H cells and approx8% for the 293/HER2-O cells. Similar activation of acidification by anti-HER2 antibody treatment was also observed for SK-BR-3 and SK-OV-3 cells (data not shown). These results indicate that the HER2 expressed in transfected cells is functionally coupled to cellular second messenger pathways.


Figure 5: Lack of acidification response by HER2-transfected CHO-K1 and 293 cells to heregulin-alpha. HER2-transfected CHO-K1 and 293 EBNA cells were prepared and loaded into Cytosensor capsules as described under ``Materials and Methods.'' The cells were monitored in the Cytosensor until acidification rates stabilized, and then media containing 100 ng/ml heregulin-alpha or 2 µg/ml anti-HER2 monoclonal antibody were switched into the fluid stream for 15 min. The cells treated with anti-HER2 were immediately exposed to 4 µg/ml anti-mouse IgG for an additional 15 min. A, CHO/HER2-H cells were exposed to heregulin (bullet) or media alone (circle). B, CHO/HER2-H cells were exposed to anti-HER2 (bullet) or control IgG1 (circle) followed by anti-IgG. Parental CHO-K1 cells were also exposed to anti-HER2 () or control IgG1 (box) followed by anti-IgG. C, 293/HER2-O cells were exposed to heregulin (bullet) or media alone (circle). D, 293/HER2-O cells were exposed to anti-HER2 () or control IgG1 (box) followed by anti-IgG. Parental CHO-K1 cells were also exposed to anti-HER2 () or control IgG1 (box) followed by anti-IgG. Due to the presence of a high concentration of buffer salts in the anti-HER2 antibody preparation, the apparent acidification rates were depressed by approximately 12% any time the preparation was present in the chamber.



Increased Acidification Response to Heregulin in HER2-transfected MCF-7 cells

To test the hypothesis that heregulin responsiveness is dependent upon a tissue-specific component in addition to HER2, a mammary carcinoma cell line expressing low levels of HER2, MCF-7, was transfected with pCEP/HER2 and selected for clones overexpressing HER2. One clone of HER2-transfected MCF-7 (MCF/HER2-A) that was found to overexpress the HER2 mRNA and p185 protein was compared with the parental MCF-7 cells with respect to response to heregulin-alpha in the Cytosensor. The MCF/HER2-A clone gave an enhanced response to heregulin and a 20% increase in the acidification rate when treated with 50 ng/ml of heregulin-alpha (Fig. 6A), and the parental cell line again gave approximately a 6% increase in rate when treated with the same amount of heregulin (Fig. 6B). These results indicate that the MCF-7 cells possess a component that reconstitutes the heregulin receptor in combination with the transfected HER2 molecules.


Figure 6: Increased acidification response to heregulin-alpha in HER2-transfected MCF-7 cells. Cells were prepared and loaded into Cytosensor capsules as described under ``Materials and Methods.'' A, the MCF/HER2-A cells were monitored in the Cytosensor until acidification rates stabilized, and then media containing 50 ng/ml heregulin-alpha (bullet) or media alone (circle) were switched into the fluid stream for 15 min. B, parental MCF-7 cells were also tested for response to 50 ng/ml heregulin-alpha (bullet) or media alone (circle) under the same conditions as in A.




DISCUSSION

Ligand binding/cross-linking and tyrosine phosphorylation assays have been used widely to study the interaction between heregulin and HER2 in breast cancer cells(1, 2, 18) . In the present study, we have employed extracellular acidification measurements to quantitate the specific response of HER2-expressing mammalian cells to recombinant heregulin-alpha. It has been demonstrated that ligand-receptor interactions generally result in the activation of second messenger pathways that trigger increases in extracellular acidification as measured by a microphysiometer(19, 20) . Earlier studies showed that tyrosine kinase-dependent EGF receptor signaling activates increased extracellular acidification(19, 23) . Our results here show that human mammary carcinoma cells MDA-MB-453 and SK-BR-3 respond strongly and rapidly to heregulin stimulation by increasing their extracellular acidification rate, whereas MCF-7 cells, another mammary carcinoma cell line, only give a very weak response. The heregulin responsiveness of these mammary carcinoma cells correlates well with HER2 expression levels as determined by immunohistochemical staining and Northern blot and Western blot analyses. This correlation does not, however, apply to CHO-K1 and 293-EBNA cells stably transfected with HER2 cDNA or to an ovarian carcinoma cell line SK-OV-3, which naturally expresses high levels of HER2. These cells fail to respond to heregulin at all. In concordance with our results, a previous biochemical study demonstrated heregulin cross-linking to HER2 and heregulin-activated tyrosine phosphorylation of p185 in human tumor cells of breast, colon, and neuronal origin but not in ovarian cells that express the receptor in natural abundance nor in transfected ovarian cells and fibroblasts overexpressing the receptor.(14) . Taken together, these results indicate that heregulin activation of cells is dependent on the presence of a tissue-specific component in addition to HER2, which is absent in ovarian cells. This hypothesis is further supported by our finding that the mammary carcinoma cell line MCF-7 that is transfected with HER2 cDNA and selected for overexpression gives an enhanced acidification response to heregulin.

It is conceivable that the additional component required for heregulin responsiveness may be a tyrosine kinase receptor closely related to HER2, such as HER3 or HER4, and that the heregulin pathway may be mediated by receptor cross-talk or heterodimerization. This possibility is suggested by several studies that have shown EGF receptor-mediated tyrosine phosphorylation of HER2 (26, 27, 28, 29) in transformed rodent and human cell lines. EGF receptor and HER2 have also been shown to act synergistically to trigger transformation of rodent fibroblasts (30) by a mechanism that may involve EGF-induced heterodimerization of EGF receptor and HER2(31) . Using the sensitive RT-PCR method and Western blotting, we detected expression of HER3 at much greater levels in mammary carcinoma cells than in ovarian cells. In contrast, the ovarian cell line SK-OV-3 was the only one tested that expressed significant levels of HER4 message, although the HER4 protein was not detectable by Western blotting. The correlation between extracellular acidification and other data suggests that cells expressing both HER2 and HER3 respond to heregulin stimulation, whereas those expressing HER2 and low levels of HER4 do not. This is in agreement with a recent study in which co-expression of HER2 and HER3 in COS-7 cells reconstituted high affinity heregulin binding and heregulin-induced tyrosine phosphorylation(17) . Our findings, on the other hand, are in contrast to an earlier study that indicated that HER4 mRNA was undetectable in ovarian cells but was expressed in mammary carcinoma cells(6) . The same group also reported data indicating that HER4 is a co-receptor required for heregulin-mediated signal transduction (15) using receptor-transfected cells. This may be the case for cells expressing high levels of the HER4 protein.

In summary, our results indicate that HER2 expression alone does not impart heregulin responsiveness. Moreover, based on our evidence, cells naturally co-expressing HER2 and HER3 respond to heregulin; we speculate that HER3 is the receptor for heregulin, which is in agreement with the results of Carraway et al.(16) and Kita et al.(32) . Ligand-driven heterodimerization of HER3 and HER2 may form a ligand-receptor complex that in turn triggers the heregulin signal transduction pathway as foreseen by Peles et al.(14) and proposed by Sliwkowski et al.(17) . In subsequent experiments, we have co-transfected HER2 and HER3 cDNA into CHO cells and reconstituted the acidification response to heregulin in these cells, (^2)which validates our results with the mammary carcinoma cells. Our present study also indicates that HER4 does not play a role in heregulin-mediated signal transduction in the mammary carcinoma cells tested.


FOOTNOTES

*
This work was supported in part by Defense Advance Research Projects Agency Contract MDA972-92-C-0005. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Mayo Medical School, Rochester, MN 55905.

To whom correspondence should be addressed. Tel.: 408-747-3513; Fax: 408-747-3601.

(^1)
The abbreviations used are: EGF, epidermal growth factor; RT, reverse transcription; PCR, polymerase chain reaction.

(^2)
S. D. H. Chan, K. S. Fok, and H. Garrett Wada, manuscript in preparation.


ACKNOWLEDGEMENTS

-We thank Margaret Hirst for expert advise on Cytosensor experiments and Wally Parce for a critical reading of the manuscript.


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