©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mat-8, a Novel Phospholemman-like Protein Expressed in Human Breast Tumors, Induces a Chloride Conductance in Xenopus Oocytes (*)

(Received for publication, June 21, 1994; and in revised form, October 18, 1994)

Briggs W. Morrison (§) J. Randall Moorman (1) Gopal C. Kowdley (1) Yvonne M. Kobayashi (2) Larry R. Jones (2) Philip Leder (¶)

From the  (1)Department of Genetics, Harvard Medical School, Howard Hughes Medical Institute, Boston, Massachusetts 02115, the Departments of Internal Medicine (Cardiovascular Division) and Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, and the (2)Departments of Internal Medicine and Biochemistry, The Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis, Indiana 46202.

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We recently identified a novel 8-kDa transmembrane protein, Mat-8, that is expressed in a subset of murine breast tumors. We have now cloned a cDNA encoding the human version of Mat-8 and show that it is expressed both in primary human breast tumors and in human breast tumor cell lines. The extracellular and transmembrane domains of Mat-8 are homologous to those of phospholemman (PLM), the major plasmalemmal substrate for cAMP-dependent protein kinase and protein kinase C in several different tissues. PLM, which induces chloride currents when expressed in Xenopus oocytes, contains consensus phosphorylation sites for both cAMP-dependent protein kinase A and protein kinase C in its cytoplasmic domain. In contrast, the cytoplasmic domain of Mat-8 contains no such consensus phosphorylation sites and is, in fact, unrelated to the cytoplasmic domain of PLM. RNA blot analysis reveals that Mat-8 and PLM exhibit distinct tissue-specific patterns of expression. We show that expression of Mat-8 in Xenopus oocytes induces hyperpolarization-activated chloride currents similar to those induced by PLM expression. These findings suggest that Mat-8 and PLM, the products of distinct genes, are related proteins that serve as Cl channels or Cl channel regulators but have different roles in cell and organ physiology.


INTRODUCTION

We recently identified a set of genes that are expressed in murine breast tumors initiated by the transgenic oncogenes c-neu or v-Ha-ras, but not in tumors initiated by the oncogenic transgene c-myc(1) . One of the markers identified, Mat-8 (Mammary tumor, 8 kDa), is a novel 8-kDa protein with a single membrane-spanning domain. The expression of Mat-8 cannot be induced in c-myc initiated tumors by the introduction of an activated neu oncogene nor can its expression be inhibited in neu-initiated tumors by the introduction of c-myc(1) . We therefore believe that Mat-8 is a marker of a cell type preferentially transformed by Neu or Ras oncoproteins. Since this marker may also be relevant to human breast cancer and point to cells at risk for the development of malignancy, we have undertaken to determine whether Mat-8 is expressed in human breast tumors and to determine what its function might be.

As an initial approach toward understanding the function of Mat-8, we searched the sequence data base and found that the extracellular and transmembrane domains of Mat-8 are homologous to those of phospholemman. Phospholemman (PLM) (^1)is a small transmembrane protein from canine cardiac sarcolemmal vesicles that has been purified, sequenced, and cloned(2) . PLM is the major plasmalemmal substrate for cAMP-dependent protein kinase A and protein kinase C in heart and other tissues and contains consensus phosphorylation sites for both of these protein kinases in its cytoplasmic tail(2) . It subsequently was shown that PLM expression in Xenopus oocytes injected with PLM RNA caused the appearance of hyperpolarization-activated chloride currents(3) .

Given the homology between PLM and Mat-8, we hypothesized that Mat-8, like PLM, may be involved in regulating chloride ion flux. Indeed, we found that injection of murine Mat-8 RNA into Xenopus oocytes induces a hyperpolarization-activated chloride current similar to that induced by PLM expression. RNA blot analysis shows different tissue-specific patterns of expression of murine Mat-8 and PLM, suggesting that their physiologic roles may differ. RNA blot analysis demonstrates that Mat-8 is expressed in both primary human breast tumors and in breast tumor cell lines.


MATERIALS AND METHODS

Library Construction, Screening, and cDNA Sequencing

A cDNA encoding murine Mat-8 (1) was excised from the Bluescript plasmid by digestion with XhoI and EcoRI, labeled with [alpha-P]dCTP using the random hexamer technique(4) , and used as a probe to screen a human breast cDNA library (Clontech Catalogue No. HL1037b). Filters were hybridized in 10% dextran sulfate, 48% formamide, 4.8 times SSC (1 times SSC is 0.15 M sodium chloride, 0.015 M sodium citrate), 20 mM Tris, pH 7.5, 1 times Denhardt's reagent, 20 mg/ml sheared herring sperm DNA with 1 times 10^6 cpm/ml of probe and washed in 2 times SSC, 0.1% SDS at 55 °C for 1 h. Positive clones were plaque-purified, and phage DNA was prepared using standard techniques(5) . The insert of a single clone was subcloned into the EcoRI site of pBluescript SK vector (Stratagene), and double-stranded DNA was sequenced with a dideoxy sequencing kit (Pharmacia Biotech Inc.).

The sequence of this initial human clone showed clear homology to murine Mat-8. We next generated a cDNA library from the human breast tumor cell line SKBR-3 using the -ZAP II vector and cDNA cloning reagents from Stratagene. Approximately 10^5 unamplified phage were screened using the human Mat-8 clone that was isolated from the Clontech library. In screening the SKBR-3 library, filters were hybridized as above but washed in 0.2 times SSC, 0.1% SDS at 65 °C for 1 h. Multiple positive isolates were plaque-purified, and phagemid DNA was prepared using the Stratagene rescue protocol. Restriction enzyme analysis revealed three classes of clones of differing size. The complete sequence analysis of two independent clones representing the smallest-sized insert was determined from both strands, and limited sequence analysis of the other two classes of clones was also performed.

RNA Analysis

Total RNA was isolated from various mouse tissues and human breast tumor cell lines as described by Chirgwin et al.(6) . Poly(A) RNA was selected using oligo(dT)-cellulose (Pharmacia Biotech Inc.). RNA blot analysis was performed according to the procedures of Thomas (7) with transfer to Genescreen membranes (DuPont NEN).

The breast tumor cell lines MCF-7, SKBR-3, MDA 231, MDA 468, MDA 453, MDA 435, MDA 436, ZR75-1, BT-474, AU565, and 2MT-2 are established cell lines available from the ATCC and were generously provided to us by Dr. Ruth Sager and Dr. Ruth Lupo. RNA from 16 different primary human breast carcinomas was kindly provided by Dr. Dennis Slamon. All primary tumors were infiltrating ductal carcinomas. The 16 patients included both node-positive and node-negative patients. Only the primary tumor, and not metastatic deposits in lymph nodes, was available. Preparation of RNA from these human tumors has been described(8) . Briefly, between 0.5 and 1.0 g of primary tumor was frozen, and a portion of the tissue was ground to powder with a mortar and pestle in liquid nitrogen. A portion of the tissue powder was extracted for RNA. Histologic examination of representative sections prepared from the same frozen tissue revealed that tumor cells comprised at least 70% of the specimen with the remainder of the specimen being primarily noncellular stroma with some vascular and inflammatory cells. Thus, the RNA analyzed is derived primarily from the malignant cells. Clinical follow-up of the 16 patients was not available.

The ribosomal L32 probe used in RNA blot analysis was derived by PCR using T3 and T7 primers that flank the insert of the Bluescript plasmid containing the ribosomal L32 gene(9) . The murine Mat-8 probe was obtained by digesting a plasmid containing the murine Mat-8 cDNA (1) with EcoRI and XhoI. A cDNA encoding murine PLM was obtained by screening a mouse heart cDNA library (Clontech Catalogue No. ML1015a) with a dog PLM probe. (^2)Sequence from the Clontech murine PLM probe was used to generate synthetic oligonucleotides that were used to PCR additional murine PLM cDNA clones. (^3)The products of PCR were cloned into the plasmid pCR 1000 (Invitrogen) and sequenced. The PLM insert was excised from pCR 1000 by digestion with EcoRI and KpnI and used as a cDNA probe for RNA blot analysis. Probes were labeled with [alpha-P]dCTP using the random hexamer technique and hybridized as described for phage screening. Filters were washed in 0.2 times SSC, 0.1% SDS at 65 °C for 1 h.

RT-PCR was performed by first preparing first strand cDNA from 3 µg of total RNA using the Superscript Kit from Life Technologies Inc. (Catalogue No. 18089-011). The first strand cDNA was then subject to PCR in a 40-µl reaction consisting of 5% of the first strand cDNA, 20 mM Tris-HCl, pH 8.3, 1.5 mM MgCl(2), 25 mM KCl, 100 µg/ml gelatin, 25 µM each dideoxynucleotide, 50 pmol of each primer, and 2 units of Taq polymerase (Cetus). Amplification was conducted for 28 cycles, each cycle consisting of incubations of 94 °C for 20 s, 55 °C for 30 s, and 72 °C for 45 s. Reaction products were resolved through a 2% agarose gel, stained with ethidium bromide, and photographed. Oligonucleotides used to amplify human Mat-8 were (sense) 5` GACTGGCACAGCCTCCAG 3` and (antisense) 5` TTTCTGTGCAGAGACAGG 3` yielding a 236-bp product and to amplify murine Mat-8 were (sense) 5` GGCTTTGACATGCAAGAG 3` and (antisense) 5` ACCTTCAGAGCCAGGACC 3` to yield a 316-bp product.

Xenopus Oocyte Assays

Removal of the 5`-untranslated region and use of the pSP64T vector (10) greatly improved oocyte expression of PLM(3) . We therefore removed the 5`-untranslated region of murine Mat-8 prior to expression in Xenopus oocytes. The PCR was used to modify the full-length murine Mat-8 cDNA to insert a Bgl-2 site 6 bp prior to the initiation methionine and a second Bgl-2 site 3 bp beyond the stop codon. This modified Mat-8 cDNA was cloned into the Bgl-2 site of pSP64T (10) to yield SP64T-Mat-8. The sequence of SP64T-Mat-8 was confirmed by the dideoxy sequencing method. SP64T-Mat-8 was linearized with SalI, and sense-capped mRNA transcripts were generated using SP6 polymerase and reagents from Ambion.

Our methods for oocyte isolation, RNA injection, and microelectrode voltage clamping have been described(3, 11) . Briefly, defolliculated oocytes were placed in a recording chamber (volume 3 ml) that was perfused at 3-4 ml/min with a solution containing (mM) NaCl, 150; KCl, 5; MgCl(2), 1; CaCl(2), 2; dextrose, 10; HEPES, 10 (pH 7.4). Microelectrodes (type 6010, AM-1 Systems, Inc., Everett, CA) were beveled to sharpness on an electrode grinder (Narishige EG-6, Tokyo, Japan). They were filled with 3 M KCl, and tip resistances were usually 1-3 M. The cells were voltage-clamped using a conventional two-microelectrode voltage clamp amplifier (Oocyte Clamp OC725, Warner Corp., New Haven, CT), connected to commercially available data acquisition and analysis software (Axon Instruments, Foster City, CA) and an IBM-compatible microcomputer. Two-second hyperpolarizing and depolarizing voltage steps were administered from holding potentials of -10 mV (near the resting potential) at 0.1 Hz. The currents were filtered at 50 to 100 Hz and sampled at 1 kHz. All experiments were performed at room temperature.

For the reversal potential determinations, the chamber was initially perfused at 3-4 ml/min with a solution containing (mM) NaCl, 200; KCl, 5.4; CaCl(2), 1.8; MgCl(2), 1; dextrose, 10; and HEPES, 10 (pH 7.4). The perfusate was sequentially changed to a series of solutions in which Cl was replaced by MES. The Cl concentrations were 200, 112, 64, 36, and 20 mM. The bath was connected to ground by an Ag-AgCl electrode in 3 M KCl agar. Ground potential changed by 2.6 ± 0.3 (S.D., n = 8) mV when [Cl](0) was changed from 0 to 150 mM. Currents were activated by 0.5 to 2 s steps from -10 mV to -120 to -160 mV, and the current traces during subsequent steps to -60 mV to 30 mV were fit with single exponential functions. The steady state component was neglected, and the initial amplitude extrapolated to the beginning of the tail current was plotted as a function of tail potential. Once normalized to the largest tail potential for each oocyte, the tail current amplitude serves as an estimate of P(0), the probability of channel opening during the hyperpolarization step. The voltage sensitivity of activation gating was estimated by calculating the gating valence z(12, 13)

where R, T, and F have their usual thermodynamic meanings, and V is the potential of the hyperpolarizing step. The term log P(0)/V is the slope of the plot of log P(0) as a function of voltage and is called the limiting logarithmic sensitivity(14) . We obtained this by fitting a line to the data over the range in which tail current rises from about 1% to about 10% of its maximum.

Antibody Production and Immunoblots

A peptide consisting of amino acids 2 through 17 of the extracellular domain of murine Mat-8 (see Fig. 3) was synthesized and coupled to KLH (Pierce Catalogue No. 77107). Fifty micrograms of coupled peptide were used per injection to immunize a single rabbit (Pocono Rabbit Farm). Various bleeds were tested initially for reactivity in an enzyme-linked immunosorbent assay in which the Mat-8 peptide was coupled to bovine serum albumin (Pierce Catalogue No. 77107). Reactive serum was affinity-purified using bovine serum albumin-coupled Mat-8 peptide linked to Affi-Gel.


Figure 3: Sequence alignment of human and murine Mat-8, canine PLM, and sheep Na,K-ATPase -chain. Sequences are aligned to maximize homology as determined by using the Genetics Computer Group sequence analysis software(31) . The transmembrane domain of human Mat-8 is underlined. Asterisks indicate phosphorylation sites for cAMP-dependent protein kinase A and protein kinase C present in PLM.



To demonstrate Mat-8 expression, 25 injected oocytes were homogenized in 500 µl of lysis buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 10 mM EDTA, 100 mg/ml phenylmethylsulfonyl fluoride), and 5-µl aliquots were run on a 15% SDS-polyacrylamide gel using a Tris-Tricine buffer system(15) . Cell lysates were also prepared from the mouse breast tumor cell line 16MB9a, which expresses Mat-8 mRNA at very low levels(1) , and from SMF, which expresses at least 100-fold more Mat-8 mRNA than 16MB9a(1) . SMF or 16MB9a cells (5 times 10^6) were lysed in lysis buffer, and 25-µl aliquots were analyzed on the 15% SDS-PAGE gel under reducing conditions (2% beta-mercaptoethanol). Electrophoresed proteins were transferred to Immobilon-P, and the immunoblot was performed with affinity-purified antibody. The secondary antibody was peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Catalogue No. 111-035-003) and was developed using ECL reagents from Amersham.


RESULTS

Isolation of the Human Homologue of Mat-8

Initial RNA blot analyses of RNA from human breast tumors using a murine Mat-8 cDNA as a probe gave no signal even under low stringency washing conditions. We therefore isolated cDNA clones encoding human Mat-8 by screening a human breast cDNA library with a murine Mat-8 probe and washing at low stringency. Phage DNA was prepared from one positive clone, subcloned, and partially sequenced. This cDNA was clearly homologous to murine Mat-8 (not shown). We next constructed a cDNA library using SKBR-3 poly(A) RNA as a template (SKBR-3 expresses Mat-8; see Fig. 2). The initial human Mat-8 cDNA clone was used as a probe to screen about 10^5 unamplified phage of the SKBR-3 library. Analyses of 7 clones revealed five containing inserts of about 500 bp and one each containing 1500 bp and about 3000 bp. All clones contained poly(A) tails.


Figure 2: RNA analysis of Mat-8 expression in human breast tumors. Blots were probed with a human Mat-8 cDNA probe and with a ribosomal L32 probe (9) as a control for RNA loading. A, 10 µg of total RNA extracted from 16 primary human breast tumors(8) . 28 and 18 indicate the location of 28 S and 18 S RNAs. Arrow indicates direction of migration of RNA. The ribosomal L32 hybridizing band is 0.6 kb. B, 10 µg of total RNA extracted from each human tumor cell line. Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum, 2 mML-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin, and total RNA was isolated by the method of Chirgwin(6) . Mat-8 expression was detected both by RNA blot (lane N; 0.5-kb transcript) and by RT-PCR (lane RT; 236-bp product).



Fig. 1shows the cDNA and deduced amino acid sequence of a clone containing a 509-bp insert. The nucleotide sequence of human Mat-8 is 75% homologous to murine Mat-8(1) . There is a single open reading frame of 264 base pairs flanked by 59 and 186 base pairs of 5`- and 3`-untranslated sequence, respectively. The putative initiation methionine is in a good context (16) for translational initiation. Restriction mapping and partial sequence analysis of the single clone containing a 1500-bp insert revealed that approximately 800 bp of additional 3`-untranslated sequence followed by a poly(A) tail were present exactly at the point where the poly(A) tail of the 509-bp clone begins. Thus, the 1500-bp clone simply reflects different polyadenylation usage and does not affect the open reading frame shown in Fig. 1. Note that the sequence shown in Fig. 1has an atypical polyadenylation signal, which likely accounts for the alternative usage of a more 3` polyadenylation site. The single clone containing a 3000-bp insert is identical with nucleotides 81 through 509 shown in Fig. 1, including the use of the proximal polyadenylation site. However, the sequence of the 3-kb clone diverges 5` to nucleotide 80. This alternate 5` sequence removes the initiation methionine and encodes alternative residues and two in-frame stop codons prior to residue 9. Thus, the 3-kb clone should not give rise to any Mat-8-related protein. We have not sequenced the remaining 2500 bp of the 5`-end of this 3.0-kb clone.


Figure 1: Nucleotide and deduced amino acid sequence of human Mat-8. Nucleotides are numbered consecutively at the right, and amino acids are numbered at the left. The putative leader sequence is underlined, the putative first amino acid of the mature protein was determined using the criteria of von Heijne(30) , and the putative transmembrane domain is double-underlined. Sequence surrounding the initiation methionine fits the consensus for translation initiation with a purine at position -3 (residue 57, (14) ). An imperfect polyadenylation signal is boldly underlined.



The human Mat-8 protein has a putative leader sequence (amino acids 1 through 20). The mature protein contains 67 amino acids with a calculated molecular weight of 8300. Amino acids 19-39 contain predominantly hydrophobic residues (with the exception of cysteine at residues 24 and 29) that encode the putative transmembrane domain of this protein. The extracellular portion of the protein is acidic with a pI of 5.3, while the intracellular portion is basic with a pI of 8.3.

Mat-8 Is Expressed in Human Breast Tumors

RNA blot analysis was performed using the 509-bp human Mat-8 cDNA as a probe on RNA from primary human breast tumors. Sixteen tumors were analyzed and, as is shown in Fig. 2A, all 16 expressed Mat-8. In all cases, three RNA species of 0.5 kb, 1.5 kb, and 3.0 kb were detected, with the 0.5-kb species predominating. It is likely that the 0.5-kb, 1.5-kb, and 3.0-kb cDNA clones we isolated from the SKBR-3 library represent the three mRNAs detected in the RNA blot. The apparent variation in expression of Mat-8 in the different tumors is generally due to different amounts of RNA loaded per well as revealed by comparison with the ribosomal L32 probe. The only clear exceptions to this are tumor 886, which appear to express Mat-8 RNA at higher levels, and tumor 1229, which expresses Mat-8 RNA at a low level. The cellularity and percentage of tumor cells was not significantly different in these tumors. Analysis of human breast carcinoma cell lines (Fig. 2B) revealed that Mat-8 RNA is present in 8 of 11 cell lines. Again, in all cases, three transcripts were detected, with only the predominant 0.5-kb transcript shown in Fig. 2B.

Mat-8 Is Homologous to Phospholemman

A search of the nucleotide and protein data banks (17) revealed that the extracellular and transmembrane domains of Mat-8 are homologous to those of PLM. Fig. 3shows the alignment of the mature forms of human and mouse Mat-8 and canine PLM. Like PLM, the extracellular domain of Mat-8 is acidic. The transmembrane domain of Mat-8, although conserved, is distinct from that of PLM by virtue of the presence of two cysteine residues. The cytoplasmic domain of PLM contains consensus phosphorylation sites for cAMP-dependent protein kinase A and protein kinase C (noted in Fig. 3). Note, however, the cytoplasmic domain of Mat-8 is unrelated to that of PLM and contains no consensus phosphorylation sites, although serine 69 has a nearby lysine and may be a substrate for PKC.

The nucleotide and protein data base search also revealed some homology between PLM and Mat-8 and the -subunit of Na,K-ATPase isolated from sheep kidney and subsequently cloned from rat, mouse, and cow kidney (18, Fig. 3shows sheep sequence). The similarity is confined to a portion of both the extracellular and transmembrane regions (Fig. 3). The functional significance, if any, of the homologous amino acids is unknown.

Mat-8 Expression Induces a Hyperpolarization-activated Chloride Current in Xenopus Oocytes

Injection of PLM RNA into Xenopus oocytes gives rise to hyperpolarization-activated chloride currents(3) . Given the homology between Mat-8 and PLM, we were interested to know whether Mat-8 also induces a chloride channel conductance. We therefore inserted the protein-coding region of Mat-8 into the translation vector pSP64T (10) and transcribed Mat-8 mRNA. Injection of Mat-8 RNA into Xenopus oocytes resulted in expression of Mat-8 protein as detected by immunoblot (Fig. 4). Under voltage clamp, oocytes injected with Mat-8 RNA demonstrated large inward currents during hyperpolarizing voltage steps (Fig. 5B). Endogenous chloride currents (12) were small in these oocytes (Fig. 5A). The threshold for activation of the current was about -90 mV (Fig. 5C). Both the current-voltage relationship and the voltage sensitivity of activation (1.9 charges, inset to Fig. 5C) of oocytes injected with Mat-8 are similar to currents induced by PLM expression(3) .


Figure 4: Detection of Mat-8 protein in Xenopus oocytes injected with Mat-8 RNA. One percent of homogenates from 25 oocytes either uninjected (lane 1) or injected with Mat-8 RNA (lane 2) were electrophoresed, transferred to Immobilon-P, and incubated with affinity-purified antibodies to the extracellular domain of Mat-8 as described under ``Materials and Methods.'' Lysates from the mouse breast tumor cell lines 16MB9a, which expresses Mat-8 mRNA at low levels, and SMF, which expresses high levels of Mat-8 mRNA(1) , were also analyzed. Molecular mass markers are shown at left. An approximately 8-kDa protein is indicated by the arrow.




Figure 5: Membrane currents in oocytes injected with Mat-8 RNA. Panels A and B, whole oocyte currents elicited using microelectrode voltage clamp steps from -10 mV to -30 to -160 mV. Averaged whole oocyte currents in 7 uninjected oocytes from 4 frogs (A) and in 7 sibling oocytes injected with Mat-8 RNA (B). Panel C, current-voltage relationship for the 14 oocytes from 4 different frogs used to create the traces in panels A and B; bars are S.D. Inset, plot of normalized tail current amplitude at 40 mV as a function of the voltage of a 2-s conditioning prepulse. The straight line yields an estimated gating valence of 1.9 charges. Panel D, the reversal potential of the current, measured from tail currents, as a function of [Cl](0). The straight line is the Nernst relationship for a perfectly selective Cl current.



The data were derived from experiments with 7 uninjected oocytes and 7 oocytes expressing Mat-8. The oocytes were obtained from 4 frogs, and the results are representative of more than 20 oocytes expressing Mat-8. The analysis was similar to our previous characterization of PLM-induced hyperpolarization-activated currents in Xenopus oocytes(3, 12) .

To determine the ionic selectivity of the current, we measured the dependence of the reversal potential on [Cl](0). According to the Nernst equation, the reversal potential varies logarithmically with the transmembrane concentration gradient of the permeant ion. For a perfectly selective channel, E changes by 58.5 mV for a 10-fold change in the concentration gradient. Fig. 5D shows a semilogarithmic plot of E as a function of [Cl](0). Since E depends on [Cl](0), we can conclude that the current is carried largely by Cl. The straight line plots the Nernst equation for a perfectly selective Cl channel. Since the observed data deviate at low [Cl](0), we can conclude that this current is not perfectly Cl-selective. Similar conclusions were reached for hyperpolarization-activated Cl currents in Xenopus oocytes induced by PLM expression (3) or for endogenous currents(12) . In those currents, the Hofmeister series of selectivity among anions was observed. In other systems conducted under bionic conditions, anion-selective currents show limited but measurable cation permeation as well, with P/P values ranging between 0.15 and 0.3(19, 20) .

Tissue Distribution of Mat-8 and Phospholemman mRNA

RNA blot analysis was performed with RNA isolated from various mouse tissues (Fig. 6). The RNA blot was probed with a murine Mat-8 cDNA probe, a murine PLM probe, and a ribosomal protein L32 probe (9) as a control for RNA loading. The murine PLM probe detects a single 0.7-kb transcript that is expressed at high levels in the heart, skeletal muscle, and liver, with low levels of expression in the breast, brain, lung, stomach, and colon. This pattern of expression is in agreement with the previously described pattern of expression of canine PLM(2) . The murine Mat-8 probe detects only a 0.6-kb transcript (1) (as opposed to human Mat-8, Fig. 2). Murine Mat-8 is expressed at high levels in the SMF cell line, uterus, stomach, colon, and at lower levels in virgin breast, ovary, lung, small intestine, and thymus. In contrast to PLM, Mat-8 is not expressed in liver, heart, or skeletal muscle, even when assayed by RT-PCR.


Figure 6: Tissue-specific expression of Mat-8 and PLM. Ten micrograms of total RNA isolated from each organ of a virgin FVB/N mouse and the SMF cell line were electrophoresed, transferred to Genescreen, and probed sequentially with a murine Mat-8 probe, a murine PLM probe, and a ribosomal L32 probe (lanes N) as described under ``Materials and Methods.'' Expression of Mat-8 was also assayed by RT-PCR (lane RT). Mouse Mat-8 detects a 0.6-kb transcript, and mouse PLM detects a 0.7-kb transcript. The RT-PCR product of Mat-8 is 316 bp. After a 10 times longer autoradiographic exposure, the 0.7-kb band of PLM is clearly visible in virgin breast, brain, lung, stomach, and colon.




DISCUSSION

We have found that transgenic murine breast tumors initiated by neu or v-Ha-ras oncogenes express high levels of Mat-8 mRNA whereas tumors initiated by c-myc do not(1) . Moreover, transfection experiments support the notion that Mat-8 is a marker of the cell type that is preferentially transformed by neu or v-Ha-ras(1) . In this report we have isolated a cDNA encoding the human version of Mat-8 and have shown that it is expressed in human breast tumors. Our analysis of primary tumors is limited by the fact that the RNA analyzed represents a mixture of malignant cells as well as stromal, endothelial, and inflammatory cells. However, the tumors we have analyzed were predominantly composed of malignant cells with most of the remainder of the specimen being stroma that is relatively poor in cellularity and makes only minimal contributions to total RNA(8) . In addition, we have also assayed human tumor cell lines and found that many of such pure populations of cells express Mat-8. The fact that tumor cell lines express Mat-8 further supports our conclusion that most of the hybridization signal we see from primary tumors comes from the malignant cells.

Our analysis of transgenic murine mammary tumors revealed a correlation between the expression of Mat-8 and specific initiating events in mammary tumorigenesis(1) . Exploring a similar correlation in human breast tumors is not possible because, except for inherited breast tumor syndromes, we do not know which of the many mutational events detected in a human tumor is the initiating event. Nonetheless, we can ask whether Mat-8 expression correlates with, for example, overexpression of neu. In our small sample of tumors, it does not. That is, even though SKBR-3 and BT-474 are known to overexpress Neu (21, 22) and MCF-7, and MDA 468 cells express normal levels of Neu, all four cell lines express Mat-8. Similarly, the primary human tumors we have analyzed all express Mat-8, but only a subset overexpress Neu(8) . The single primary tumor that expresses Mat-8 at a higher level (Fig. 1) expresses a normal level of Neu. We should emphasize, however, that the power to detect such associations in such a small sample is quite limited. It is likely that immunohistochemistry will allow us to explore these correlations in a large number of tumors as well as to determine whether Mat-8 expression provides information regarding clinical outcome or response to therapy.

The passage of chloride across the cell membrane is involved in a number of cellular functions including transepithelial transport, stabilization of the membrane potential, signal transduction, and the regulation of cell volume(13, 23, 24) . Chloride channels may be activated by changes in voltage or by ligand-receptor interactions, and the type of regulation is likely to be specific to the cellular function being controlled. The molecular characterization of chloride channels is therefore central to understanding a wide variety of physiologic processes.

A number of proteins capable of inducing chloride currents in Xenopus oocytes have been cloned and characterized. Some, such as ClC-2(23) , are ubiquitously expressed and likely play some central housekeeping role in cellular physiology. Others, such as ClC-1 (24) , have quite limited expression patterns and likely play a more specialized role. Interestingly, although ClC-1 and CLC-2 are large proteins with numerous transmembrane domains(23, 24) , PLM expression in Xenopus oocytes also induces chloride currents(3) , yet PLM contains only 72 amino acids and possess only one transmembrane domain(2) .

The data presented here show that Mat-8 expression, like PLM, induces a hyperpolarization-activated chloride current in Xenopus oocytes. However, the two proteins display distinct tissue-specific patterns of gene expression that at once suggest distinct cellular functions. The expression of Mat-8 in the breast, lung, stomach, and colon, coupled with the ability of Mat-8 protein to induce chloride channel activity, suggests that this protein may be involved in regulation of transepithelial transport in tissues containing absorptive or secretory epithelium. Conversely, the high level expression of PLM in the heart and skeletal muscle suggests other roles. In addition, PLM appears to be regulated by phosphorylation, whereas there is no evidence for such regulation of Mat-8.

We have not determined the role of Mat-8 in cell physiology, but its induction of Cl currents in Xenopus oocytes hints that its function may lie in regulation of transmembrane ion flux. Like PLM, Mat-8 can be viewed as either a Cl channel or a Cl channel regulator, and electrophysiologic measurements of whole oocyte Cl currents may not be able to distinguish between these two possibilities(12) . The identity of the better-studied I or min K molecule, which also has a single transmembrane domain and induces slow ion currents in Xenopus oocytes, faces a similar uncertainty. The main evidence that I is a channel are the findings that mutant I molecules induce currents with altered selectivity (25) and kinetics(26) . Similarly, PLM mutants also induce currents with altered kinetics (3) and voltage sensitivity of activation(12) . The main evidence that I is a channel activator is the finding that it can induce not only depolarization-activated K currents but also hyperpolarization-activated Cl currents(27) , a property not shared by PLM(27) . In our opinion, the function of these molecules, which may constitute a new superfamily of membrane transport proteins, is not yet settled. It will be interesting to pursue lipid bilayer reconstitution experiments with purified PLM (28, 29) and Mat-8.

Why is Mat-8 expressed in breast tumors? It is clear that Mat-8 is not expressed by all cell types ( Fig. 2and Fig. 6, (1) ) and hence does not perform some housekeeping function common to all cells. We have previously demonstrated (1) that Mat-8 is a marker of a specific cell type preferentially transformed by the action of the Neu and Ras, but not Myc, oncoproteins during murine mammary carcinogenesis. There are two possible explanations for this observed tropism. One is that Mat-8 is a required component of Neu and Ras transformation, and, therefore, Mat-8 expression and function are critical to the maintenance of the transformed state. Conversely, it may be that the oncogenic target cell expresses Mat-8 as a characteristic feature of a secretory epithelial cell prior to transformation and that the expression of Mat-8 is simply maintained after transformation. We have not yet distinguished between these two possibilities, but we hope to do so by inhibiting Mat-8 expression or function in tumors that express Mat-8. If Mat-8 function is a critical component of certain classes of oncogenic events, then inhibitors of Mat-8 activity may be deleterious to the survival of certain cancer cells. We are now in the position to begin to understand the cellular function of the Mat-8-induced chloride current and to determine the effects of modulating Mat-8-induced channel activity on breast tumor biology.


FOOTNOTES

*
This work was supported by National Institutes of Health grants (to B. W. M., L. R. J., and J. R. M.), the Herman C. Krannert Fund (to L. R. J.), an American Heart Association Established Investigator Award (to J. R. M.), and the Howard Hughes Medical Institute. 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: the Dana-Farber Cancer Institute, Division of Hematologic Malignancies, Boston, MA 02115.

To whom correspondence and reprint requests should be addressed. Tel.: 617-432-7667; Fax: 617-432-7663.

(^1)
The abbreviations used are: PLM, phospholemman; PCR, polymerase chain reaction; RT, reverse transcription; bp, base pair(s); kb, kilobase(s).

(^2)
L. R. Jones, unpublished data.

(^3)
L. R. Jones and S. Cala, unpublished data.


ACKNOWLEDGEMENTS

We are grateful to Dennis Slamon for providing RNA from primary human breast tumors, Ruth Lupo and Ruth Sager for providing human breast tumor cell lines, and Nissim Benvenisty and the other members of the Leder Laboratory for advice and encouragement. We would like to thank Angela Hoover Morrison for reading the manuscript.


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