(Received for publication, June 21, 1994; and in revised form, October 18, 1994)
From the
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.
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) ()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.
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
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
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.
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. ()Sequence from the Clontech murine PLM probe was used to
generate synthetic oligonucleotides that were used to PCR additional
murine PLM cDNA clones. (
)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
[
-
P]dCTP using the random hexamer technique
and hybridized as described for phage screening. Filters were washed in
0.2
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, 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.
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, 1; CaCl
,
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, 1.8;
MgCl
, 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
]
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
, 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/V is
the slope of the plot of log P
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.
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
10
) were lysed in lysis buffer, and 25-µl aliquots were
analyzed on the 15% SDS-PAGE gel under reducing conditions (2%
-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.
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.
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.
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]
. 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]
. 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
]
. Since E
depends on [Cl
]
, 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
]
, 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) .
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.
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.