(Received for publication, August 10, 1994; and in revised form, December 20, 1994)
From the
Neutral endopeptidase is a cell surface zinc metallopeptidase that regulates the action of a variety of physiologically active peptides. The enzyme exhibits a wide tissue distribution, being most abundant in kidney and lung. Three rat neutral endopeptidase cDNAs with unique 5`-untranslated sequences were isolated. Distribution of the corresponding mRNAs in rat tissues was analyzed by RNase protection assays and by in situ hybridization. In kidney, the type 2b transcript was the major species. In lung and testis, type 1 and type 2b transcripts were expressed in approximately equal amounts, while in brain and spinal cord the type 1 mRNA was the major transcript. These findings were extended by in situ hybridization studies. All three mRNAs were expressed in the proximal tubule of the kidney, with the type 2b transcript giving the strongest signal. In the frontoparietal cortex, expression of the neutral endopeptidase mRNA subtypes was cell- and region-specific. The type 1 transcript was localized to neurons, type 2b mRNA was not detectable, while type 3 mRNA was localized to the oligodendrocytes of the corpus callosum. These results clearly demonstrate that expression of the three neutral endopeptidase mRNAs can be regulated in a cell-specific manner.
Neutral endopeptidase 24.11 (NEP, ()neprilysin,
enkephalinase, EC 3.4.24.11) is a zinc metallopeptidase that regulates
the physiological action of a variety of peptides by lowering their
extracellular concentration available for receptor binding. NEP
exhibits a broad tissue distribution with kidney containing the highest
enzyme concentration(1) . High levels of NEP are also found in
intestine, adrenal glands, and lung, with lower levels found in brain
and spinal cord. The physiological role of NEP largely depends on
substrate availability. For example, in kidney and vascular
endothelium, NEP may play a role in regulating atrial natriuretic
peptide levels(2, 3) . In the central nervous system,
the enzyme appears to regulate enkephalin-induced
analgesia(4) ; in the lung, substance P levels are regulated by
the enzyme(5) , while in endometrium, the enzyme may regulate
endothelin levels(6) . Human NEP is identical to CD10, the
common acute lymphoblastic leukemia antigen(7, 8) ,
which is expressed at the pre-B cell stage but is absent at more mature
B cell stages, suggesting that NEP may be involved in B cell
development and/or differentiation.
NEP gene expression can be stimulated in human granulocytes by tumor necrosis factor, methionine enkephalin, or substance P(9) . Similarly, NEP activity in human endometrium is cyclically induced as serum progesterone levels rise and fall(6) , while enzyme expression on CD10 positive cells as well as on rabbit mammary epithelial and fibroblast cells is reduced by treatment with phorbol diester(10, 11) .
Shipp et al.(12) demonstrated the presence of three human NEP cDNAs each containing unique 5`-untranslated sequences but a common coding sequence. Comparison of various NEP cDNA sequences revealed that this gene has been highly conserved among mammalian species(13) . Isolation of the human gene for NEP by D'Adamio et al.(14) led to the finding that the three mRNA species were derived from two noncoding exons separated by an intron of 600 bp. The type 1 mRNA was generated from the most upstream exon, exon 1, while the other two mRNAs were produced by alternative splicing of exon 2.
These data suggest that the NEP gene can be regulated in a tissue-specific manner. However, regulatory elements in this gene have not been characterized, nor has the tissue distribution of the alternatively spliced mRNAs been studied. Here we report the structure of the 5`-noncoding region of the rat NEP gene, the description of a previously unidentified 5`-noncoding exon, and the demonstration of tissue- and cell-specific expression of NEP mRNAs .
To make a cDNA probe that spans the type 1 exon and the first coding exon, the oligonucleotides MSA (see below) and EPR2 were used as primers for PCR. The synthesized PCR product was subcloned and sequenced as described above.
Total RNA and
poly(A) RNA isolated were fractionated by
electrophoresis on a formaldehyde (1% agarose) gel. The fractionated
RNA was transferred to a nylon membrane (Schleicher & Schuell) and
UV cross-linked. The membrane was prehybridized at 55 °C in the
buffer recommended by the manufacturer for 1.5 h and then hybridized to
the appropriate cRNA probe overnight. The membrane was washed 3 times
with 1
SSPE (0.15 M NaCl, 1 mM EDTA, and 10
mM NaH
PO
), 0.5% SDS buffer at 65
°C for 20 min and then with 0.1
SSPE, 0.5% SDS buffer at 60
°C for 20 min.
The cRNA probes specific for each noncoding exon as well as a
cRNA common probe, containing 163 bp of coding sequence between EcoRV and PvuII sites, were transcribed in vitro from the T7 promoter site (antisense orientation) or the T3
promoter site (sense orientation) in pBluescript using CTP-biotin
(nonradioactive RNA labeling system, Life Technologies, Inc.). The
biotin-labeled probes were purified using centrifugation columns (1100
g, 4 min), ethanol precipitated, and rehydrated with 2
hybridization buffer (Life Technologies, Inc.) to a
concentration range of 0.25-1.25 µg/ml.
Slide-mounted
tissue sections were covered with denatured probe solution
(0.1-0.5 µg/ml). Slides were coverslipped, sealed using
rubber cement, and placed in a humid chamber (42 °C, 18 h). The
slides were then removed from the hybridization solution, and the
rubber cement and coverslips were carefully removed by briefly dipping
in 0.2 SSC. The slides were rinsed in 0.2
SSC (3
times), washed in 0.2
SSC (22 °C, 15 min, 2 times), and
then placed in humid chambers and covered with blocking solution (22
°C, 15 min) (nonradioactive detection kit, Life Technologies,
Inc.). The blocking solution was drained, and streptavidin-alkaline
phosphatase conjugate was placed onto the tissue sections. The slides
were incubated (22 °C, 15 min) in a humid chamber and then washed
in TBS (22 °C, 15 min, 2 times) and then in alkaline substrate
buffer (22 °C, 5 min, 1 time). Slides were placed in a nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate/alkaline substrate
buffer mixture (Life Technologies, Inc.) and incubated until chromogen
reaction occurred (37 °C, 2 h). The chromogen development was
stopped by rinsing the slides in distilled water several times and then
processed through a dehydrating alcohol gradient (50, 75, 90, and 100%)
and covered with Crystal Mount (Fisher). Tissue sections were examined
and photographed using brightfield optics on a Nikon Microphot
microscope.
Previous studies on the human NEP gene led to the
identification of three distinct mRNA species derived from two
noncoding exons each containing unique 5`-untranslated sequences and a
common coding sequence. These were designated as types 1, 2a, and
2b(14) . Using PCR and the RACE technique, we isolated three
rat NEP cDNAs that corresponded to the type 1 and type 2b cDNAs of the
human gene and a unique cDNA that we have designated as type 3 (Fig. 1). The type 1 cDNA, previously identified by Malfroy et al.(18) from rat brain, was prepared by PCR from
rat kidney cDNA as described under ``Materials and Methods.''
Type 2b and type 3 cDNAs were obtained as RACE products from kidney
cDNA. The type 3 cDNA, which was not previously identified, is the
product of a downstream noncoding exon, exon 3 in the rat gene, see
below. A cDNA clone containing the type 3 noncoding exon was previously
isolated in this laboratory. ()Another mRNA, the type 2a
mRNA, was observed in the human Nalm-6 lymphoblastic leukemia cell line (14) and is derived from the same noncoding exon as the type
2b mRNA. This mRNA was not detected in any of the rat tissues used in
this study.
Figure 1: 5`-Untranslated sequences of rat NEP cDNAs and their alignment to human CD10/NEP cDNAs. The underlined common sequence of 13 bases, including the start codon ATG, is from the first coding exon. Type 1 5`-untranslated sequence of rat brain NEP cDNA was reported by Malfroy et al.(1988). Type 1, type 2a, and type 2b 5`-untranslated sequences of human CD10/NEP cDNAs were reported by D'Adamio et al. (1989).
In order to verify the existence of these mRNAs and to
determine their origin, we isolated and analyzed a rat NEP genomic
clone. cRNA probes specific for each of the noncoding exons were
synthesized and utilized to screen a rat genomic library (see
``Materials and Methods''). A positive recombinant phage,
which hybridized to all three noncoding exon probes, as well as a probe
containing the first coding exon sequence, was isolated. This DNA clone
was digested with either HindIII or XbaI, and the DNA
fragments were subcloned into pBluescript. In Fig. 2A is
shown a restriction map of the 5`-region of this genomic clone. The
organization and alternative splicing of these upstream exons is shown
in Fig. 2B. These exons were identified by comparing their
sequences with the corresponding cDNA sequences. The localization of
exon 1 and exon 2 in the rat gene is similar to that found in the human
gene (D'Adamio et al., 1989). These two exons are
separated by an intron of 600 bp. The type 2a cDNA sequence is
found upstream to the type 2b sequence in exon 2, but as shown below
the alternative splicing that occurs in human Nalm 6 cells apparently
does not occur in rat tissue. The third noncoding exon was identified
downstream of exon 2. This downstream exon, exon 3, is separated by an
intron of
3.5 kilobases from exon 2 and an intron of 155 bp from
the first coding exon in the gene, exon 4.
Figure 2: A, restriction map showing HindIII and XbaI sites in the 5`-region of the rat NEP gene. Exons1, 2, and 3 are 5`-noncoding exons. Exon4 is the first coding exon. See text for details about the size of each intron. B, four types of NEP mRNAs were generated through the ligation of different noncoding exons to the common coding exon and alternative splicing. Type 1, 2b, and 3 were found in rat tissues and type 1, 2a, and 2b were found in human Nalm-6 cells.
In an attempt to
determine the pattern of expression of each of these mRNA subtypes, RNA
probes specific for the rat type 1, type 2b, or type 3 mRNAs, as well
as a full-length rat NEP cRNA probe, were hybridized to total or
poly(A) RNA from rat tissues. As shown in Fig. 3A, a major band of 3.5 kilobases and two minor
bands of 6.6 and 2.7 kilobases, resulting from multiple polyadenylation
signals(18) , were observed for the RNA hybridized to the
full-length probe. The major band (3.5 kilobases) was also observed for
all three specific probes (Fig. 3B), suggesting that
alternative splicing of 5`-noncoding exons is not related to the
selective termination at multiple polyadenylation sites. These results
further show that NEP mRNA is very abundant in kidney but less abundant
in lung. Although the band intensity for the short specific probes is
weaker than that for a long NEP cRNA probe, the type 2b probe generates
a stronger signal than either the type 1 or the type 3 probe,
indicating that in kidney the type 2b mRNA is the major species.
Figure 3:
Northern blot of rat kidney and lung NEP
RNA. A, hybridization of the full-length neprilysin cRNA probe
to RNA. 20 µg of total RNA (lanes1 and 3) or 5 µg of poly(A) RNA (lanes2 and 4). B, hybridization of three
specific RNA probes (type 1, 2b, and 3) to RNA blots. Five µg of
poly(A)
cellular RNA from tissues as indicated was
analyzed with each probe. 3
10
cpm/ml probe was
added to the hybridization buffer. The size of fractionated RNA is
estimated by the RNA size standards.
To
overcome the difficulties in using short probes for quantifying rare
transcripts, we performed RNase protection assays to further analyze
these alternatively spliced NEP transcripts.
[P]UTP-labeled RNA probes, which were
complementary to the 5`-noncoding exon sequence (68, 100, and 100 bp
for exon 1, 2b, and 3, respectively), and the first coding exon
sequence (141 bp) were hybridized to total tissue RNA (Fig. 4A). This permitted us to compare the abundance
of a specific mRNA type with total mRNAs in the same sample. The
protected region of each type mRNA are 209, 241, and 241 bp for type 1,
type 2b, and type 3, respectively, while all other mRNAs would show up
as a 141-bp product. Fig. 4B shows the results of
typical experiment. The relative abundance of each type of NEP mRNA
transcript was quantified using a radioanalytic imaging system (Ambis
System Inc.), with the data summarized in Table 1. The results
demonstrate that the proportion of a particular NEP transcript is
tissue-dependent. In kidney, where the highest overall NEP mRNA level
is found, the type 2b mRNA is the most abundant species. In lung, type
2b and 3 mRNAs are fairly abundant, but expression of type 1 is greatly
depressed. Significantly, in whole brain and spinal cord, type 1 mRNA
is the major species.
Figure 4: A, probe designs used in RNase protection assay. Type 1, 2b, and 3 RNA probes contain specific sequences of 68, 100, and 100 bp from 5`-noncoding exon. All three probes contain the common sequence of 141 bp from the first coding exon. B, ribonuclease protection assay of total cellular RNA from kidney (a), lung (b), testis (c), brain (d), and spinal cord (e) with three specific RNA probes (type 1, 2b, and 3). Thirty µg of total RNA from each tissue were loaded in each lane. As indicated by arrows, the protected region of each type mRNA are 209, 241, and 241 bp for type 1, type 2b, and type 3, respectively. The 141 bp band is formed between the probe and all of the other neprilysin mRNAs (see text for details).
Attempts to detect the type 2a mRNA by RNase protection experiments were unsuccessful. To determine whether this mRNA was actually synthesized in rat kidney, lung, or brain, we employed reverse transcriptase PCR. For this procedure, we used the primer E2aC, 5`-GACCCTGCGAGTGATGTCAG-3` (see Fig. 1, Rat 2a sequence), which is complementary to a region that would be found in both the type 2a and 2b mRNAs (see Fig. 2B), in conjunction with a primer, EPR1 (see ``Materials and Methods''), which is complementary to the first coding exon. If the type 2a mRNA exists in any of these tissues, a PCR product of 168 bp would be formed (Fig. 5, top). As an internal control, a 326-bp band corresponding to the type 2b mRNA would be formed (Fig. 5, top). As shown in Fig. 5(bottom), only the 326 bp band was observed, indicating that the sequence found in the type 2a mRNA is part of the type 2b sequence but that the type 2a transcript does not exist at a detectable level in these tissues. The finding of a weak 326-bp PCR product from brain under the same experimental conditions is consistent with the low level of type 2b mRNA in brain found in the RNase protection assay.
Figure 5: Top, illustration of type 2a and type 2b PCR products resulting from using a type 2a primer, E2aC, and primer EPR2 to the common sequence. Bottom, PCR products for kidney (lane1), lung (lane2), and brain (lane3 (bottom)). The 123-bp ladder marker is shown in lane4. Only the type 2b PCR product was detected (326 bp), indicating the absence of the type 2a cRNA in these tissues.
Specific cell populations in the rat kidney, which contained either the type 1, type 2b, or type 3 mRNA transcripts, were further identified by in situ hybridization analysis. In the kidney, specific hybridization with the rat type 1, type 2b, and type 3 mRNA subtypes was detectable (Fig. 6). The type 2b mRNA gave the highest levels of hybridization (Fig. 6B), comparable with that of the common cRNA probe (Fig. 6D). Type 2b mRNA was clearly localized with a strong hybridization signal to the proximal tubules of the kidney (Fig. 6B). No labeling of the glomeruli, juxtaglomerular apparatus, or distal tubules was observed. The type 1 (Fig. 6A) and type 3 (Fig. 6C) transcripts were of lower relative abundance than the type 2b transcript, but were nonetheless clearly detectable and also specifically localized to the proximal tubules of the kidney. No labeled kidney cells were detected using sense orientation riboprobes for the type 1, type 2b, or type 3 transcripts.
Figure 6: Brightfield photomicrographs of rat kidney cells containing either the type 1 (A), type 2b (B), type 3 (C), or coding sequence probe (D) mRNA transcripts. Nonisotopic in situ hybridization analysis was performed as described under ``Materials and Methods'' using CTP-biotin-labeled riboprobes. Specific hybridization signal (darkcells) to the rat type 1, 2b and type 3 mRNA subtypes was localized to the proximal tubules of the kidney. Calibrationbar = 0.1 mm.
Cell
populations in rat brain containing either the type 1, type 2b, or type
3 mRNA transcripts were identified. In the rat frontoparietal cortex, a
diversity of hybridization patterns were observed (Fig. 7). The
type 1 transcript was localized to neurons and was uniformly
distributed across all cortical lamellae (Fig. 7A).
Type 2b was not present in the frontoparietal cortex (Fig. 7B) but was present in neurons of many other
brain regions, such as the ventral striatum. ()Type 3 was
prominent in the myelinated areas of the brain and localized to the
oligodendrocytes of the corpus callosum with scattered cells localized
throughout the frontoparietal cortex (Fig. 7C). The
common rat NEP cRNA probe labeled a diverse assortment of neuronal and
glial cell populations in the rat frontoparietal cortex (Fig. 7D). Tissue sections hybridized with sense
orientation probes for either the type 1, type 2b, or type 3
transcripts did not demonstrate labeled cells in any brain region.
Figure 7: Brightfield photomicrographs of cell populations in rat frontoparietal cortex containing either the type 1 (A), type 2b (B), type 3 (C), or coding sequence probe (D) mRNA transcripts. Nonisotopic in situ hybridization analysis was performed as described under ``Materials and Methods'' using CTP-biotin-labeled riboprobes. The specific hybridization signal (darkcells) for the type 1 transcript was localized to neurons scattered across all cortical lamellae (A). Type 2b was not present in frontoparietal cortex (B). Type 3 was prominent in myelinated tracts of the brain and in scattered cells throughout the frontoparietal cortex (C). The coding sequence probe labeled a diverse assortment of neuron and glial cell populations (D). Calibrationbar = 0.1 mm.
Three rat NEP mRNAs containing unique 5`-untranslated sequences were identified. Two of these mRNAs, rat type 1 and 2b, are homologues of human CD10/NEP mRNA types 1 and 2b, respectively. These are derived from noncoding exons that are highly conserved with 75% sequence identity between the rat and human genes. The alternative splicing of human exon 2, seen in Nalm 6 cells, was not observed in the rat tissues examined in this study. We were able to identify a new transcript, rat type 3 mRNA, the transcription of which was initiated from a third downstream noncoding exon. All of these noncoding exons are ligated to the first common exon, which contains the start codon and 10 bases of 5`-untranslated sequence, indicating that all of the transcripts encode the identical protein. Tissue-specific expression of these transcripts may provide a mechanism to regulate enzymatic activity in different tissues.
The data obtained in this study are
consistent with, but do not prove, that multiple promoters are involved
in regulating expression of the NEP gene. Two promoters in the
-amylase gene are involved in the tissue-specific expression in
the parotid gland and the liver of mouse (19) . Aromatase
cytochrome P-450 expression is regulated by tissue-specific promoters
for the production of a single protein(20) . A mechanism for
producing diversified proteins from a single gene is also employed by
the contractile protein genes through alternative
splicing(21) .
RNase protection assays and in situ hybridization experiments show that the level of expression of the
three NEP transcripts varies in different tissues. In kidney, where the
highest overall NEP activity is found, the type 2b transcript is
predominantly expressed. In brain, where the NEP level is relative low,
the type 1 transcript appears as the major species. In situ hybridization has revealed regulation of NEP gene expression at
the regional and cellular level. Although all three NEP mRNAs are
expressed in the same cells in the kidney, a different situation occurs
in brain tissue where specific regional distribution of the mRNA
transcripts was observed. For example, the type 2b transcript was not
found in the frontoparietal cortex but was localized to other regions
of the brain such as the ventral striatum. The regional
distribution of transcripts implies an important role for NEP mRNAs in
brain development. Moreover, differential localization to various brain
regions indicates that the various transcripts may be involved in the
regulation of specific promoter/receptor coupling. In the ventral
striatum, given the high levels of opioid receptors
present(22) , regulation of enkephalin levels by the 2b
transcript may be a unique mechanism for regulating endogenous opioid
peptides. In addition to regional specificity, the brain also displays
cellular specificity. That is, the type 3 mRNA was predominate in
myelinated pathways throughout the brain. The other transcripts were
clearly localized to neuronal populations. Thus, the three mRNAs
derived from the NEP gene can be differentially expressed in a regional
and cell-specific manner, possibly representing an important mechanism
for regulating enzyme levels in different cell types in the brain.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U18272 [GenBank]and U18273[GenBank].