(Received for publication, June 15, 1995; and in revised form, July 13, 1995)
From the
The synovial fluid or group II secretory phospholipase A (sPLA
) has been implicated as an important agent
involved in a number of inflammatory processes. In an attempt to
determine the role of sPLA
in inflammation, we set out to
generate sPLA
-deficient mice. During this investigation, we
observed that in a number of inbred mouse strains, the sPLA
gene was already disrupted by a frameshift mutation in exon 3.
This mutation, a T insertion at position 166 from the ATG of the cDNA,
terminates out of frame in exon 4, resulting in the disruption of the
calcium binding domain in exon 3 and loss of both activity domains
coded by exons 4 and 5. The mouse strains C57BL/6, 129/Sv, and B10.RIII
were found to be homozygous for the defective sPLA
gene,
whereas outbred CD-1:SW mice had variable genotype at this locus.
BALB/c, C3H/HE, DBA/1, DBA/2, NZB/B1N, and MRL lpr/lpr mice had a normal sPLA
genotype. The sPLA
mRNA was expressed at very high levels in the BALB/c mouse small
intestine, whereas in the small intestine of the sPLA
mutant mouse strains, sPLA
mRNA was undetectable. In
addition, PLA
activity in acid extracts of the small
intestine were approximately 40 times higher in BALB/c than in the
mutant mice. Transcription of the mutant sPLA
gene resulted
in multiple transcripts due to exon skipping. None of the resulting
mutant mRNAs encoded an active product. The identification of this
mutation should not only help define the physiological role of
sPLA
but also has important implications in mouse
inflammatory models developed by targeted mutagenesis.
Secretory nonpancreatic phospholipase A (sPLA
) (
)has been implicated as one of the
important pathogenetic agents in both local and systemic inflammatory
conditions (for reviews, see (1, 2, 3) ).
Levels of circulating sPLA
in both adult (4) and
juvenile (5) rheumatoid arthritis as well as in septic shock (6) and multiorgan failure (7) correlate positively
with disease severity and poor prognosis. sPLA
gene
expression has also been shown to be induced by various inflammatory
stimuli, like endotoxin, IL-1
, TNF-
, and
IL-6(1, 2) . Nevertheless, the exact mode of action of
sPLA
and the role it plays in these diseases is not very
well understood. sPLA
catalyzes the release of fatty acid
from the sn-2 position of phospholipids producing free fatty
acid and lysophospholipid. When the fatty acid is arachidonic acid,
which is usually esterified at this position in phospholipids, it can
lead to the production of the various leukotriene and prostaglandin
proinflammatory mediators. The other product of this reaction,
lysophospholipid, can also be metabolized into a potent inflammatory
mediator (platelet-activating factor). However, since sPLA
has very little preference for the type of fatty acid in the sn-2 position, it is not known to what extent the
proinflammatory activity of sPLA
is mediated through the
release of arachidonic acid and its subsequent metabolism into
proinflammatory mediators. One approach to determine the role of
sPLA
in these various disease states would be to develop
potent specific inhibitors of this enzyme. Recently, there have been a
number of sPLA
inhibitors
reported(8, 9, 10, 11, 12, 13) .
However, none of these inhibitors have been able to convincingly
determine the role sPLA
plays in inflammation. In an
attempt to circumvent this problem and investigate the role of
sPLA
in inflammation, we initiated studies to generate mice
with a targeted disruption of the sPLA
gene. Provided, the
mutation was not lethal, sPLA
-deficient mice could be used
in inflammatory models to determine the contribution of sPLA
in the inflammatory process.
During this investigation, we
observed that the sPLA gene, cloned from a 129/Sv-derived
genomic library, already contained a natural mutation. This mutation
causes a disruption in the reading frame of exon 3 resulting in an
inactive gene product. This paper describes the nature of this
mutation, its consequence, and its prevalence in a number of inbred
mouse strains.
Figure 3:
Genomic analysis of the mouse sPLA gene. A, Southern blot analysis of BamHI-digested mouse genomic DNA from the mice strains shown
and the ES cell line D3 (129/Sv-derived) was hybridized with the the
3`-end HindIII probe of
3 (Fig. 1). The two
C57BL/6 lanes represent DNA isolated from two separate animals. The
BALB/c genomic DNA was from Clontech. All other genomic DNAs were
prepared. B, analysis of PCR clones of exon 3 from the mice
indicated. Exon 3 of the sPLA
gene was amplified from mouse
genomic DNA as described under ``Materials and Methods'' and
cloned into the pCR II vector. Four clones from each amplification were
digested with EcoRI, which releases the inserted DNA and with BamHI. Lane6 from the amplification of CD-1
genomic DNA contains two exon 3 fragments, one that is digested by BamHI and the other that is not.
Figure 1:
Genomic organization of the
mouse sPLA gene. Restriction map and localization of the
sPLA
gene in the
3 and
6 sPLA
genomic
clones are illustrated. The exons are denoted by numbers below the corresponding box. The additional BamHI site in exon
3 of
3 is shown, and the HindIII genomic fragment from
the 3`-end of
3 used as the probe for Southern blotting is underlined.
Figure 2:
DNA sequence of the mouse sPLA gene. The sequence shown is from
6, with the mutation in
exon 3 removed, in order to show the correct translation of the gene.
Highlighted within the sequence are the putative TATA box, which is overlined; the five exons and the deduced amino acid sequence,
which are underlined; the BamHI site in exon 3, which
contains the T insertion mutation is in boldface and has an asterisk below it; and a putative polyadenylation site, which
is in boldface. The TATA box, exon 1, and the polyadenylation
site are based on the structure of the rat sPLA
gene(23, 24) .
However, between the two mouse sPLA
genomic clones
there appeared to be a BamHI polymorphism. When
3 was
digested with BamHI, two DNA fragments hybridized with the
sPLA
cDNA probe, a 5.7-kb 5`-fragment and a 600-bp
3`-fragment representing the end of this clone. Based on the sequence
from the sPLA
gene in
3, this BamHI site
occurs in exon 3. This appears to be a conserved sequence since this BamHI site is also present in exon 3 of the rat and human
genes. A similar BamHI digest of
6 or of embryonic stem
cell genomic DNA resulted in a single 8.2-kb fragment. An explanation
for this discrepancy is that
6 originated from the stem cell,
whereas
3 was from contaminating STO feeder cell genomic DNA. The
STO feeder cells are mouse fibroblasts derived from the inbred Swiss
strain SIM(25) . Subsequent sequencing of the sPLA
gene in
6 revealed that the BamHI site in exon 3
was lost because of an insertion of an extra T residue (GGATCC
GGATTCC). This insertion not only would disrupt the BamHI
recognition sequence, but it would also cause a frameshift in the
expressed mRNA. The resulting sPLA
enzyme would have a
disrupted calcium binding domain and terminate out of frame in exon 4,
losing both the exon 4 and 5 sequences that make up the active site
(see Fig. 5). This sPLA
enzyme would be totally
inactive since both the calcium and catalytic activity domains are
absolutely required for activity. This result would indicate that the
full-length sPLA
gene in
6 isolated from the ES stem
cell genomic library codes for an inactive gene product.
Figure 5:
DNA
sequence of wild-type and mutant sPLA cDNAs. The three
sPLA
cDNAs isolated from the CD-1
sPLA
-deficient mouse are identified as follows: ImsPLA
, contains the complete
sPLA
coding region, but it has the T insertion (asterisk) at position 166 from the ATG; X3sPLA
has exon 3 deleted; X4sPLA
has exon 4 deleted. The BALB/c
sPLA
cDNA, which codes for an active enzyme, is identified
as MsPLA
. The ATG initiation codon and
the termination codons for each sequence are shown in boldface. The position of the exon sequences within the cDNA
sequence are underlined. Two polymorphisms at positions 101
and 231 between the BALB/c and CD-1 sPLA
cDNA sequences
were found and are underlined.
There are
at least four possible explanations for this result. First, the
mutation was a sequencing error (this is unlikely since this region was
sequenced with a number of different primers on both strands, and in
addition this sequence is not cleaved by BamHI); second, the
mutation was generated during cloning; third, it could be an artifact
of the ES cell line; and fourth, the mutation originated from the
129/Sv mouse strain from which the ES cells were originally derived. If
this last possibility was correct, then there already existed a mouse
strain that had a natural disruption of the sPLA gene.
To confirm the Southern blotting result and prove that the loss of the BamHI site in exon 3 was due to the T insertion, exon 3 was amplified from each of these mice strains, cloned, and sequenced. An example of this analysis is shown in Fig. 3B, in which exon 3 clones from these mouse strains were digested with BamHI. The C57BL/6 exon 3 clones were not cleaved by BamHI, whereas all of the BALB/c exon 3 clones contained the BamHI site and gave the appropriate cleavage products. The CD-1 exon 3 clones, as expected, consisted of both types. Analysis of the exon 3 DNA sequences revealed that 129/Sv, C57BL/6 and the mutant allele in the CD-1 all had the T insertion mutation.
We have also
analyzed the sPLA gene structure for a number of additional
mouse strains, and the results are presented in Table 1. Most of
the mouse strains tested had a homozygous wild-type sPLA
BamHI digestion pattern. The heterozygosity of the
B6/D2F1 mice would indicate that the DBA/2 mouse strain has the
wild-type sPLA
gene, since C57BL/6 is homozygous for the
sPLA
mutation. The congenic strain B10.RIII has the mutated
sPLA
BamHI digestion pattern, which would mean
that the C57BL/10 mouse strain also has the mutated sPLA
gene. This is very likely since C57BL/6 and C57BL/10 are
substrains of C57BL. Based on the known genealogy (26) of
inbred mouse strains, it is possible that 129/Sv and C57BL could have
originated from a common ancestor that passed on the sPLA
mutation. The origin of the other mouse strains listed in Table 1are much further removed from these two strains.
Figure 4:
sPLA mRNA expression in mouse
small intestine. A, Northern blot analysis of total RNA
isolated from the mouse small intestine. The RNA was isolated from
control and LPS-treated animals. The 129/Sv mice were injected
interperitoneally as follows, and 5 h later the tissue was removed. Lane1, control injected with PBS; lane2, injected with 5 µg of LPS; and lane3, injected with 10 µg of LPS. The BALB/c and C57BL/6
mice were injected intravenously as follows, and 18 h later, the tissue
was removed. Lanes4 and 7, controls
injected with PBS; lanes5 and 8, injected
with 300 µg of LPS; and lanes6 and 9, injected with 500 µg of LPS. Total RNA from rat
calvarial osteoblasts that had been treated with IL-1
and
TNF-
(induces sPLA
mRNA) (48) was run as a
control for hybridization. The blot was first hybridized with a mouse
sPLA
cDNA probe and then reprobed with a human
glyceraldehyde 3-phosphate dehydrogenase probe. B, RT-PCR
amplification of wild-type and mutant sPLA
cDNAs. Total
RNAs isolated from the small intestine were used in RT-PCR reactions to
amplify the cPLA
cDNA. Lane1, BALB/c; lane2, CD-1 sPLA
-deficient mouse that
was treated with carrageenan/LPS as described under ``Materials
and Methods''; and lane3, rat osteoblast. The
RT-PCR products were analyzed on a 2% low melt agarose
gel.
The high level of sPLA mRNA expression in the
mouse intestine would suggest that this tissue should also be a good
source for the sPLA
enzyme. In fact the enzyme responsible
for the majority of the acid-stable PLA
activity in the
BALB/c small intestine was recently purified and shown to be sPLA
(32) . Therefore, the acid-extractable PLA
activity in the small intestine of BALB/c and C57BL/6 mice was
determined. The PLA
activity in the BALB/c control
intestine (126, 594 units/ml) was found to be at least 40 times higher
than in the C57BL/6 control intestine (3,031 units/ml). The small
amount of PLA
activity remaining in the C57BL/6 intestine
is most likely due to one or a combination of the other low molecular
weight secretory phospholipases A
, which are also
acid-stable. Treatment of the mice with LPS did not increase the level
of PLA
activity in either the BALB/c or C57BL/6 intestine,
which was consistent with the lack of induction by LPS on sPLA
mRNA levels in this tissue. The reason for the lack of induction
of sPLA
expression by LPS in the intestine is not clear,
since LPS or endotoxin has been shown to greatly induce sPLA
expression in the rat(33) . One likely explanation is
that the level of sPLA
expression is already so high in the
BALB/c small intestine that it would be difficult to see any amount of
induction in this tissue. In other tissues like the aorta and in the
small intestine of mice with the sPLA
mutation, sPLA
mRNA can be induced by LPS (see below).
In order to prove that
the BALB/c sPLA mRNA codes for an active sPLA
enzyme and the mutant sPLA
gene codes for an inactive
form, both mutant and BALB/c sPLA
cDNAs were cloned by
RT-PCR. We had found that it was possible to induce expression of the
sPLA
mRNA in mice carrying the mutated sPLA
gene if extreme conditions are used for the induction. When mice
are first pretreated with carrageenan and then injected 24 h later with
LPS there is a tremendous enhancement of LPS-induced TNF production and
mouse mortality by endotoxin shock(19) . Under these
conditions, sPLA
mRNA can be detected in the small
intestine by Northern blot analysis and in the aorta by RT-PCR (not
shown) of mice with the mutated sPLA
gene. It should be
noted that BALB/c mice do not survive this treatment; they die shortly
after the carrageenan injection. We have used small intestine total RNA
prepared from a CD-1 mouse that was homozygous for the sPLA
mutation (-/-CD-1) (verified by BamHI
genomic Southern blotting) and treated in the above fashion and from
BALB/c for RT-PCR to amplify and clone both the mutant and wild-type
mouse sPLA
cDNAs. The PCR products from these
amplifications are shown in Fig. 4B. Both the BALB/c
and rat osteoblast (control) RNA produced the correct size 595-bp
PLA
cDNA product (Fig. 4B), which could
also be digested by BamHI (not shown). The sPLA
cDNA products from amplification of total RNA isolated from the
-/-CD-1 mouse (or from any of the mutant sPLA
mice, 129/Sv, C57BL/6) gave multiple sPLA
cDNA
products (Fig. 4B, lane2) that could
not be digested by BamHI (not shown). Both the BALB/c
sPLA
cDNA and the mutant sPLA
cDNA RT-PCR
products were cloned and sequenced and are shown in Fig. 5. The
two more strongly amplified products from the -/- mouse
were the full-length sPLA
cDNA with the T insertion
(ImsPLA
) and a sPLA
cDNA with exon 3 removed
(X3sPLA
). A third sPLA
cDNA with exon 4 spliced
out was also found (X4sPLA
). These sPLA
cDNAs
would most likely code for inactive enzymes since they all have
disrupted calcium binding and catalytic activity domains. Both the
mutant and wild-type sPLA
cDNAs were cloned into the
eukaryotic expression vector pSG5 and transiently transfected into COS7
cells. 48 h after transfection, PLA
activity in the media
of the transfected cells was determined. Cells transfected with any of
the mutant sPLA
cDNAs did not release any detectable
PLA
activity into the media, confirming that mice strains
carrying the mutant sPLA
gene do not express an active
sPLA
enzyme. In contrast, the media of cells transfected
with the BALB/c sPLA
cDNA contained greater than 3,000
units/ml of PLA
activity, confirming that the BALB/c
sPLA
gene codes for an active enzyme.
In this study we have shown that the murine sPLA gene is naturally disrupted in a number of inbred strains. This
discovery came about following initial experiments to disrupt
sPLA
by targeted mutagenesis in order to derive a
sPLA
mutant model. It has been recognized that to increase
the efficiency of gene targeting in ES cells, the targeting vector must
be derived from DNA that is isogenic to the cell line(20) .
While screening a genomic DNA bacteriophage library prepared from the
ES cell CC1.2, a BamHI restriction polymorphism was discovered
between an isogenic sPLA
clone and a clone that originated
from the contaminating STO feeder cells. This polymorphism was due to
the insertion of a thymidine residue in exon 3 of the ES cell gene. The
resulting transcribed mRNA would contain a frameshift mutation and code
for an inactive enzyme, since both the calcium binding and catalytic
activity domains encoded by exons 3, 4, and 5 were absent. The CC1.2 ES
cell line, having been derived from the mouse strain 129/Sv, suggested
that a sPLA
mutant already existed in mouse populations,
while STO cells, having been derived from an inbred Swiss
line(25) , indicated that the gene was not mutant in all
strains. A survey of inbred mouse stains demonstrated that the
sPLA
gene is disrupted in 129/Sv and C57BL/6 but not in
BALB/c. It is likely that this mutation would have been overlooked had
the polymorphism between CC1.2 and STO not been observed. Therefore, it
may be advisable that before attempting to generate targeted mutations
in mice, one should first check the expression of the particular gene
in a panel of inbred strains. Based on the above observations, there
exists the possibility that ``knockout'' mice with no overt
phenotype, may have had the gene of interest already disrupted.
The
loss of the sPLA gene appears not to have a detrimental
effect on the viability of these mice strains. C57BL/6 is one of the
mostly widely used mouse strains and accounts for more than 14% of
studies in which an inbred strain is used(34) . Thus, the
sPLA
gene does not appear to play a critical role in the
development of the animal, nor does it appear to have an effect on
fertilization as has been suggested(35, 36) . In fact,
there is no obvious phenotype for the sPLA
mutation.
However, with the identification of sPLA
-deficient mouse
strains, the physiological role of sPLA
and its involvement
in inflammatory conditions can now be addressed.
The high levels of
sPLA present in inflammatory synovial fluids of rheumatoid
arthritis patients was suggestive of a proinflammatory role for
sPLA
in this disease(1, 2, 3) .
The levels of circulating sPLA
also correlated very well
with disease severity in both adult and juvenile rheumatoid
arthritis(4, 5) . However, the function of sPLA
in rheumatoid arthritis is unknown. It has been suggested that
its involvement in rheumatoid arthritis may be to potentiate an already
established inflammatory process(1) . The mouse has been used
extensively as a model for arthritis. Knowing the sPLA
genotype of several of the inbred mouse strains, we attempted to
see if there was any correlation between mouse strain, the
functionality of the sPLA
gene, and the arthritic model. In
antigen-induced arthritic models, both sPLA
-deficient and
sPLA
wild-type mouse strains are susceptible to arthritis.
The sPLA
-deficient strains C57BL/6 and B10.RIII have been
used in methylated bovine serum albumin (37) and collagen (38) induced models of arthritis, respectively, whereas, BALB/c
mice appear to be susceptible to proteoglycan-induced
arthritis(39) . Conversely, mouse strains such as DBA/1 and MRL lpr/lpr, which have a wild-type sPLA
gene
structure, are susceptible to a spontaneous occurring
arthritis(40, 41) . From such results, it would seem
that sPLA
is not required for the initiation of arthritis,
but it is possible that it may have a more subtle role perhaps in
disease severity or prolongation, which is more difficult to decipher
from these studies. Attempting to draw conclusions about the role of
sPLA
in arthritis from previous studies is complicated by a
number of factors, such as the genetic variability of each inbred
strain and the complex nature of these arthritic models. A similar
problem arises when trying to apply this type of analysis to other
sPLA
proinflammatory activities such as its involvement in
eicosanoid production and in septic shock. The best approach to address
these issues and obtain a better understanding of the role of
sPLA
in these diseases would be to generate sPLA
wild-type and sPLA
-deficient mice with the same
genetic background. Such mouse strains could then be used to reevaluate
the role of sPLA
in these disease models.
Given the
potential role of sPLA in inflammation, our results suggest
that caution must be exercised in generating genetic models for
inflammation based on the application of targeted mutagenesis in the
mouse. The targeted disruption of a gene is always carried out in ES
cells derived from the 129/Sv mouse strain, while the mutation is bred
into either the 129/Sv or C57BL/6 background, both of which bear the
natural sPLA
gene disruption. Such mice will have mutations
in two proinflammatory genes, which would greatly complicate the
interpretation of results. Therefore, it is perhaps advisable that gene
targeting experiments of proinflammatory genes be conducted in a
genetic background where the sPLA
gene is intact.
A
number of other functions have also been attributed to
sPLA. Mulherkar et al.(27, 28, 29, 30, 31) have
localized the murine sPLA
to the Paneth cell of the BALB/c
small intestine and have proposed that sPLA
acts as an
enhancing factor for epidermal growth factor binding to cells, thus
playing a role in cellular proliferation within the small
intestine(27, 28, 29, 30, 31) .
Another group has recently purified sPLA
from the BALB/c
small intestine and has shown it to have significant bactericidal
properties(32) . They suggest that the anti-microbial activity
of sPLA
protects the small intestine from microbial
infection. Consequently, sPLA
-deficient mice might be
expected to be more susceptible to small intestine infections and have
problems in regulating the high cell turnover that occurs in this
tissue. However, sPLA
-deficient mouse strains appear not to
have any gross observable differences in the physiology of the small
intestine(34) . This would suggest that either the mice survive
without these sPLA
activities or that perhaps the minimal
PLA
activity still present in the intestine of the mutant
mice is sufficient to maintain homeostasis. There are at least three
other low molecular weight sPLA
s identified in humans and
rodents. These are the pancreatic PLA
and two recently
cloned PLA
s, one of which appears to be localized in the
heart (42) and the other in the testes(43) . Whether or
not these other secretory PLA
s compensate for the loss of
sPLA
activity in these mice remains to be determined.
Nonsense or frameshift mutations have been shown to cause reduced
steady-state levels of mRNA (for review, see (44) and (45) ). The insertion of a single T residue into exon 3 of the
mouse sPLA gene demonstrates this quite effectively. In
mice carrying this mutation, no sPLA
mRNA was detectable by
Northern blot analysis in the small intestine, whereas in wild-type
animals, it is present at very high levels. However, the mutant
sPLA
mRNA can be induced if more extreme conditions are
used for the induction. In this case, multiple transcripts of the
mutated sPLA
gene, due to exon skipping, are observed.
These transcripts code for inactive sPLA
enzymes. Normally
exon skipping has been observed in genes with mutations involving the
splice donor or acceptor sequences. The mutation in the sPLA
gene occurs -24 bp from the 5` splice site. Recently, there
have been a few reports of single base exonic mutations that result in
aberrant mRNA splicing(46, 47) . One such mutation was
identified in the mitochondrial acetoacetyl-CoA thiolase gene (T2) from
a girl with T2 deficiency(46) . This mutation occurs in exon 8,
-13 bp from the 5` splice site of the intron and causes partial
skipping of this exon. It was suggested that this mutation could result
in an altered secondary structure of the T2 pre-mRNA around exon 8 and
in this way impede normal splicing. Thus it is possible that the
secondary structure of the mouse sPLA
pre-mRNA is altered
by the single base insertion mutation in exon 3.
While this
manuscript was in review, MacPhee et al.(49) published a paper describing this same sPLA mutation in inbred mouse strains. They present evidence to show
that the sPLA
gene maps to a locus, Mom1, that
modifies Min-induced tumor number in the murine small and
large intestine. They suggest that sPLA
modifies polyp
number by altering the cellular microenvironment within the intestinal
crypt. How sPLA
carries out this function remains to be
determined.
In conclusion, we have shown that the sPLA gene is disrupted in a number of common inbred mouse strains.
This could impact on gene targeting experiments since the mouse strains
129/Sv and C57BL/6 bear this mutation. Its role in arthritis is still
not clear, but it appears not to be required for the initiation of the
disease. Experiments are in progress to generate sPLA
wild-type and sPLA
-deficient mice with the same
genetic background and reevaluate the role of sPLA
in mouse
inflammatory models.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U32313[GenBank], U32358[GenBank], and U32359[GenBank].