From the
The urokinase-type plasminogen activator receptor (uPAR) gene
was disrupted in mice in order to explore the role of cell
surface-associated plasminogen activation in development and
hemostasis. Homozygous, uPAR
Extracellular proteolysis focused at the cell surface is a key
factor in both cell migration through tissue barriers and cell-mediated
modification of tissue
architecture(1, 2, 3, 4) . The
plasminogen activator/plasmin system of proteins, which includes
urokinase-type plasminogen activator (uPA),
Cell surface-associated plasminogen
activation may also play a critical role in pathological cell migration
events, including invasive dissemination of tumor cells in metastatic
cancer(1, 3, 14, 15, 16, 17, 18, 19) and smooth
muscle cell migration in atherogenesis(20) . Notably, PA and/or
PA receptor expression is consistently observed at the invasive front
of both spontaneous and experimentally-induced cancers. The biological
importance of the PA/plasmin system in malignancy is highlighted by the
fact that specific inhibitors of either plasminogen activator, plasmin
or plasminogen activator receptor, have frequently been shown to
inhibit tumor cell migration both in vitro and in
vivo(14, 15, 16, 17, 18, 19) .
The uPA receptor (uPAR) is increasingly recognized as an important
factor in focusing plasmin-mediated pericellular proteolysis. uPAR is a
55-70 kDa glycoprotein that is attached to the plasma membrane
through a post-translationally added glycosylphosphatidylinositol
anchor(21) . The protein contains three cysteine-rich domains
that are similar to elements found in the Ly-6 superfamily of
glycosylphosphatidylinositol-anchored proteins, whose members include
the murine Ly-6 antigens(22, 23) , CD59 (membrane
inhibitor of reactive lysis; Ref. 24), the squid brain glycoprotein
Sgp-2(25) , and the viral protein HVS-15(26) . The
amino-terminal domain of uPAR is known to mediate uPA
binding(27) , while the function of the two other domains
remains to be elucidated. uPAR binds uPA ligand with both high
specificity and affinity (K
Several recent reports have suggested that uPAR
may have additional roles not directly related to plasminogen
activation. For example, uPAR appears to bind the extracellular matrix
protein, vitronectin, through a region outside of the amino-terminal
uPA-binding domain(41) . This interaction was shown to influence
the adhesion properties and morphology of cultured cells even in the
absence of uPA(41) . Furthermore, uPAR appears to be required
for chemotaxis of human monocytes in vitro in a manner
independent of the uPA ligand (42). In order to clarify the
physiological and pathological roles of uPAR in vivo, we
recently isolated the murine uPAR gene (43) and now report the
use of this cloned DNA to generate uPAR-deficient mice.
Breeding studies in uPAR-deficient
mice showed that the absence of uPAR does not appear to compromise the
fertility of either males or females. The average litter size observed
in 20 litters sired by uPAR
The ability of plasmin to degrade common extracellular matrix
components and the diverse contexts in which plasminogen activators,
receptors, and inhibitors are expressed has suggested that cell
surface-associated plasminogen activation may play a critical role in
embryonic and adult tissue remodeling and cell migration. The results
of this study indicate that uPAR-mediated processes are not essential
for development, growth to adulthood, or reproduction. More than 200
uPAR-deficient mice have now been raised, 19 of which have been
monitored over a period of 6-8 months, and we have observed no
obvious abnormalities in these animals.
There is no evidence for
either residual uPAR activity or another, compensatory uPA receptor in
any of the primary tissues and cultured cells that we have analyzed
from uPAR
Interestingly, our studies indicate that uPAR is not critical to
pro-uPA activation in at least one organ system, the urogenital tract.
As typically found with normal mice(59) , most of the uPA in
urine of uPAR
The first of three
structurally-related domains found in uPAR is important for ligand
binding (27) but the function(s) of the other two domains are
still poorly defined. One intriguing possibility is that these domains
mediate binding to other ligands or cell surface components, perhaps in
concert with uPA. One cell-surface protein that interacts with uPAR is
low density lipoprotein receptor-related
protein/
One surprising
finding in this study was that the mutant uPAR allele lacking the third
exon (and containing a large HPRT minigene) generated significant
amounts of uPAR mRNA that appeared to result from the splicing of the
second and fourth exon sequences. No such 2-to-4 splicing pattern was
observed by reverse transcriptase-PCR using RNA templates from normal
cells, but this must occur in mutant transcripts at a rate that is
competitive with the rate of RNA cleavage and polyadenylation in the
intervening HPRT sequences. All of the exon-intron junctions of the
uPAR gene are of the same type (i.e. type I; occurring between
the first and second nucleotide of each codon). Thus, translation of
the mutant mRNA would remain ``in-frame'' producing a protein
product lacking the residues encoded by exon 3 (which corresponds to a
major portion of the ligand binding domain). Immunoassays using two
independent antibodies specific for mouse uPAR, including one that is
known to recognize forms of uPAR lacking domain-1(57) , have
provided no indication of a truncated form of uPAR in
uPAR
If the only essential physiological role
of uPAR is to direct uPA-mediated plasminogen activation, then the
apparently normal development, growth, and fertility of uPAR-deficient
mice is consistent with earlier findings that plasminogen-deficient (68) and uPA-deficient (60) mice complete embryonic development,
survive to adulthood, and are fertile. The most profound consequence of
plasminogen deficiency is the development of severe thrombosis and
vaso-occlusive pathologies(68) . A small percentage of
uPA-deficient mice also develop fibrin deposits, particularly in the
liver(60) . These observations reinforce the importance of
plasminogen activation in fibrinolysis and hemostasis and suggest that
uPA/uPAR may participate, along with tPA, in fibrin clearance. One
simple model for a dual role of uPA and tPA in fibrinolysis is that uPA
may participate in cell-mediated fibrinolysis via its interactions with
uPAR(3) , whereas tPA may primarily mediate cell-independent
fibrinolysis via its fibrin-binding properties(5) . This concept
is consistent with many of the kinds of cells known to express uPAR
and/or uPA, including macrophages(12, 29) ,
keratinocytes (10, 11), and capillary endothelial
cells(31, 32, 33) , which must be able to
penetrate and/or organize within fibrin matrices occurring at sites of
infection, inflammation, or trauma. If the hypothesis is correct that
uPA/uPAR-mediated plasminogen activation is critical for cellular
turnover of fibrin-rich matrices, then the phenotypic consequences of
uPAR deficiency might be most apparent in mice challenged by an added
deficit in tPA or animals challenged by infection or injury.
An
assortment of proteases, including serine-, metallo-, aspartyl-, and
thiol-proteases, appear to be necessary for the modification and
degradation of complex extracellular matrices with diverse protein
compositions(1, 2, 3, 4, 63, 64, 65) .
If the proteases employed in matrix degradation share a partial
functional overlap with regard to both sites of expression and
substrate specificity, then it follows that the loss of any one
component would not necessarily compromise overall extracellular matrix
remodeling or turnover. Some overlap in substrate specificity between
proteases is well-established. Notably, uPA and tPA share the same
substrate and partially complement one another genetically(60) .
Likewise, stromelysin 1 (MMP-3), stromelysin 2 (MMP-10), and other
metalloproteases can degrade type-IV collagen, gelatin, fibronectin,
and laminin(63) . Both plasmin and stromelysin can degrade
fibronectin and laminin, and activate
procollagenase(13, 63) . Therefore, based on the
observations made here with uPAR-deficient mice, it cannot be concluded
that the uPAR/uPA/plasmin system does not participate in general matrix
degradation in the context of developmental or adult tissue remodeling;
rather, it can only be concluded that this system is not essential for
development to term in the absence of other functional deficits or
other challenging factors. More detailed studies of
uPAR/uPA/plasminogen-deficient mice, including breeding studies with
mice lacking other extracellular proteases, should illuminate the
interplay between distinct protease systems.
A linkage between
plasminogen activation and cancer is well-established (1, 3) and
inhibitors of both uPA and uPAR have been shown to effectively suppress
tumor cell metastasis in a number of model systems (14, 17-19).
The uPA/uPAR system may be critical in tumor cell penetration of
fibrin, which is commonly deposited around solid tumors (66). However,
tumor cells may also capitalize on the ability of plasmin to degrade
other matrix proteins and activate other proteases (13). The
availability of both uPAR-deficient mice and transgenic mouse lines
genetically predisposed to develop malignant tumors (67) should
provide the means to precisely define the role of surface-associated
plasminogen activation in tumor cell dissemination.
We thank Drs. Sandra Degen and Mary Jo Danton for
their advice and critical reading of the manuscript. We are grateful to
Kenn Holmbäck and John Duffy for their expert assistance in
generating gene-targeted transgenic mice. We also thank Jean Snyder and
the personnel of the pathology laboratory for their assistance in the
histological analyses.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
mice were born
and survived to adulthood with no overt phenotypic abnormalities. There
was no indication of loss of fetal animals based on the Mendelian
pattern of transmission of the mutant uPAR gene.
uPAR
mice carried no detectable uPAR in lung,
spleen, and other tissues when measured both immunologically by Western
blot analysis and functionally by ligand cross-linking analyses. In
addition, activated peritoneal macrophages collected from
uPAR
mice failed to promote plasminogen
activation in vitro. The loss of the receptor also resulted in
a redistribution of uPA in some tissues but had no impact on pro-uPA
activation in the urogenital tract. Thus, in the absence of other
challenging factors such as infection, injury, or other functional
deficits, uPAR deficiency does not compromise fertility, development,
or hemostasis. These mice provide a means to test the proposed function
of uPA/uPAR in wound repair, atherogenesis, and tumor cell invasion in vivo.
(
)tissue-type plasminogen activator (tPA), plasminogen, and
PA- and plasminogen-specific cell surface receptors and inhibitors,
provides one proteolytic mechanism whereby cells can manipulate their
local environment(1, 2, 3) . Although one clear
substrate for this system is fibrin(5) , a number of
observations have suggested that the PA/plasmin system may play a more
general proteolytic role in matrix turnover. Notably, expression of
plasminogen activator has been documented in the context of
ovulation(6) , trophoblast invasion(6) , neuronal cell
migration(7) , angiogenesis(8, 9) , keratinocyte
migration(10, 11) , and tissue infiltration by
inflammatory cells(12) . The expression of PA and related
inhibitors and receptors is sensitive to a variety of growth factors,
cytokines, and hormones(1, 2) , factors that are likely
to regulate the timing, location, and degree of extracellular
proteolysis. A role of plasmin in extracellular matrix proteolysis is
also consistent with its ability to both directly and indirectly (e.g. through metalloprotease activation; Ref. 13) degrade
common matrix proteins.
10
to 10
M)
through the uPA ``growth factor-like'' domain (28) and
directs uPA activity to the leading edge of migrating
cells(29) . Furthermore, the receptor potentiates uPA-mediated
plasminogen activation by increasing the rate of pro-uPA activation (by
plasmin), reducing the apparent K
of uPA
for plasminogen substrate, and increasing the apparent k
for plasminogen activation(30) .
Consistent with the broad utility of plasmin-mediated proteolysis, uPAR
has been identified on many diverse types of cells, including monocytes
and macrophages, endothelial cells, smooth muscle cells, keratinocytes,
trophoblasts, and many types of tumor
cells(3, 11, 29, 31, 32, 33, 34, 35, 36) .
Interestingly, uPAR, like many other glycosylphosphatidylinositol
proteins(37) , may participate in signal transduction, including
directing uPA-dependent changes in protein phosphorylation and cell
proliferation(38, 39) . However, the mechanism by which
uPAR might alter intracellular events has not yet been established. In
addition to focusing uPA activity, uPAR also appears to be critical in
the ultimate clearance of uPA
inhibitor complexes from the cell
surface(40) .
Generation of uPAR-deficient Mice
A uPAR
targeting vector was generated using a fragment of the mouse 129 strain
uPAR gene(43) . A 5550-bp EcoRI fragment containing the
portion of the uPAR gene extending from +1080 (intron 1; converted BglII site) to +6630 (intron 3) was isolated from
uPARg-1 (43) and subcloned into a modified Bluescript
(SK+) vector (Stratagene) lacking the polylinker BamHI
site. A 2-kb ClaI cassette encoding herpes simplex
virus-thymidine kinase was inserted into the plasmid polylinker ClaI site immediately upstream of the intron 1 sequences to
provide a means of selection against non-homologous
recombination(44) . Finally, a 1-kb BamHI fragment
containing exon 3 (43) was removed and substituted with a 6-kb BamHI cassette encoding human HPRT (45) having the same
orientation as the uPAR gene elements relative to transcription.
Vector-free targeting vector DNA (40 µg) was introduced by
electroporation (800 V/cm and 200 microfarads; IBI GeneZapper) into
HPRT-deficient E14TG2a (46) embryonic stem cells (10
cells transfected in a 750-µl suspension prepared in
serum-containing medium). The electroporated cells were maintained on
mitomycin C-treated primary mouse embryonic fibroblasts (47) for
24 h and then placed in selection medium containing 0.1 mM
hypoxanthine, 16 µM thymidine, 0.4 µM aminopterin (Life Technologies, Inc.), and 2 µM ganciclovir (Syntex). Drug-resistant clones were picked after 7
days, expanded in 24-well dishes, and DNA extracts were prepared and
tested by PCR using a primer complementary to the HPRT cassette
(OligoHPRT-1; 5`-TATTACCAGTGAATCTTTGTCAGCAG-3`; Ref. 45) and a primer
complementary to a uPAR intron 3 sequence (OligoPAR-25;
5`-GAACCAGTGAGCCAACC-3`; corresponding to +6755 to +6771 in
the gene sequence; Ref. 43). Correct targeting of two PCR-positive
clones was confirmed by Southern blot hybridization analyses using exon
1- and exon 2-specific hybridization probes and genomic DNA digests
prepared with three different restriction enzymes (HindIII, PvuII, and BglII). Each clone was injected into the
blastocoel cavity of C57BL/6 blastocysts and subsequently implanted
into the uterine horns of pseudo-pregnant females(47) . Chimeric
male offspring were mated to NIH Black Swiss mice (Taconic) to generate
heterozygous offspring. These mice were then interbred to generate
homozygous uPAR
progeny.
Genotype Analysis
Genotypes of mice were
determined using tail biopsy DNA (47) of 3-week-old mice by
Southern blot or PCR analysis. In the Southern blot assays, PvuII- or BglII-digested genomic DNAs were hybridized
with either a 108-bp probe complementary to exon 2 or a 77-bp probe
complementary to exon 1. The exon 1 probe was generated by PCR using
the primer uPAR-E2-5` (5`-CCTCCCAGGGCCTGCAGTGCA-3`; corresponding
to +2640 to +2660 in the gene sequence; Ref. 43) and primer
uPAR-E2-3` (5`-GCCATTCCCGAAGCACGGTAG-3`; corresponding to
+2727 to +2747 in the gene sequence; Ref. 43). The exon 2
probe was generated by PCR using the primer uPAR-E1-5`
(5`-AGAGCAGAGGTGAAGGAAGAA-3`; corresponding to +1 to +21 in
the gene sequence; Ref. 43) and primer uPAR-E1-3`
(5`-ACACAGGTAGTCGCCAGCAAC-3`; corresponding to +57 to +77 in
the gene sequence; Ref. 43). In the PCR-based genotype assays, the
targeted uPAR allele was detected using the uPAR intron 3 primer
uPAR-I3 (5`-TCATCAGTCCTCCCTGCTAAGGGC-3`; corresponding to +5290 to
+5313 in the gene sequence; Ref. 43) and OligoHPRT-1 (see above).
The wild-type uPAR allele was detected with exon 3 primers,
uPAR-E3-5` (5`-GATGATAGAGAGCTGGAGGTGGTGAC-3`; corresponding to
+4178 to +4203 in the gene sequence; Ref. 43) and
uPAR-E3-3` (5`-CACCGGGTCTGGGCCTGTTGCAGAGGT-3`; corresponding to
+4299 to +4325 in the gene sequence; Ref. 43).
RNA Analysis
Total RNA was isolated from the
embryonic fibroblasts as described(48) . Twenty-µg samples
were fractionated by denaturing agarose gel electrophoresis,
transferred to BA85 nitrocellulose (Schleicher and Schuell), and
hybridized (49) with a P-labeled 1500-bp mouse uPAR
cDNA probe(50) . Equivalent sample loading was verified by UV
transillumination of ethidium bromide-stained ribosomal RNAs. Mouse
tissue RNA was isolated using Oligotex d(T) (Qiagen), reverse
transcribed in 30-µl reaction mixtures containing 200 ng of
poly(A)
RNA, 24 units of avian myeloblastosis
virus-reverse transcriptase (Boehringer Mannheim), and 50 µmol of
random hexanucleotide primers, and then PCR amplified using Taq polymerase (Boehringer Mannheim) and the uPAR primers,
5`-GCAGTGTGAGAGTAACCAGAGCT-3` (exon 2; +2662 to +2684 in the
gene sequence; Ref. 43) and 5`-CCACAGCCTCGGGTGTAGTCCT-3` (exon 5;
+10084 to +10105 in the gene sequence; Ref. 43).
Poly(A)
RNA isolated from PMA-treated normal mouse
skin was used as a positive control.
uPAR Immunoblot and Ligand Cross-linking
Assays
Tissues were frozen in liquid nitrogen, ground into a
powder, and Triton X-114 detergent-phase extracts prepared (1 ml of
buffer per 100 mg of powdered tissue) as described
previously(51) . Extracted proteins were fractionated by
SDS-polyacrylamide gel electrophoresis, transferred to filters, and
immunostained (51) with either a rabbit antisera raised against
uPAR domain 1 peptide (residues 26-45 using the mature protein
numbering system; Ref. 43) or domain 2 peptide (residues 126-145
using the mature protein numbering system; Ref. 43). A detailed
procedure for the cross-linking of human ATF to uPAR will be published
elsewhere.(
)Briefly, detergent-phase extracts
were combined with
I-labeled human ATF in 20-µl
reaction mixtures containing 2.5 nM ATF. The mixtures were
incubated at 4 °C for 1 h and the products covalently cross-linked
by addition of 20 mM
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 20 mM sulfo-N-hydroxysuccinimide (Pierce) with continued
incubation at room temperature for 20 min. The reaction was stopped by
the addition of 10 mM ammonium acetate. The radiolabeled
products were separated by electrophoresis on polyacrylamide (10%) gels
and detected by autoradiography. In the competition experiments,
unlabeled mouse ATF was added to a final concentration of 100 nM prior to addition of labeled ATF.
Cell-surface Plasminogen Activation
Peritoneal
cells, primarily activated macrophages, were collected from lavage
fluid prepared from mice 72 h after administration of an
intraperitoneal injection of 0.5 ml of thioglycollate medium (Difco).
The mice were sacrificed under CO narcosis and the
peritoneal cavity was flushed with 6 ml of RPMI 1640 (Life
Technologies, Inc.). The cells were washed once in RPMI 1640 and frozen
at -80 °C in RPMI 1640 containing 10% fetal calf serum and
10% Me
SO until use. 5
10
macrophages
were seeded in 96-well plates and maintained for 24 h in RPMI 1640
containing 10% fetal calf serum. The cells were washed once in RPMI
1640 and then incubated with 1 nM murine pro-uPA for 15 min at
37 °C. The murine pro-uPA used in these incubation mixtures was
purified from serum-free conditioned medium of mouse sarcoma
virus-transformed 3T3 cells(52, 53) . The cells were
washed twice in 50 mM Tris-HCl (pH 7.4) containing 100 mM NaCl and 1% bovine serum albumin, once in 50 mM Tris-HCl
(pH 7.4) containing 100 mM NaCl, and finally incubated with
human plasminogen (0.5 µM) and the plasmin-specific
chromogenic peptide substrate
H-D-Val-Leu-Lys-p-nitroaniline (0.3 mM) in
50 mM Tris-HCl (pH 7.4) containing 100 mM NaCl and
0.01% Tween 80 (54). Substrate hydrolysis was determined as A
in a Molecular Devices Thermo-Max
plate reader operating in kinetic mode at 37 °C.
Plasminogen Activator Zymography
Peritoneal lavage
fluid was collected as described above and the cell pellet and
supernatant fractions were separated by a 10-min centrifugation at 2000
g at 4 °C. The pelleted cells were lysed and
solubilized directly in polyacrylamide gel sample buffer containing 2%
SDS but lacking reducing agents. The cell-free supernatant fraction was
clarified by centrifugation for 45 min at 17,500
g at
4 °C and then combined with a 2-fold concentrate of gel sample
buffer. Protein concentrations were determined using the Micro BCA
assay (Pierce). Ten µg of cell-free supernatant protein and 25
µg of cell extract protein were analyzed by zymography using
polyacrylamide gels cast with casein and plasminogen as described
previously (55) except 10 mM EDTA was included in gel
wash solutions. Pulmonary lavage fluid was prepared from mice
anesthetized with 0.1 ml/30 g body weight of
ketamine:xylazine:acepromazine (4:1:1). A 19-gauge catheter was
inserted into the trachea and tied firmly in position with silk thread.
Cold phosphate-buffered saline (1 ml) was gently introduced into the
lungs and then withdrawn. The lavage was repeated and the withdrawn
fluids combined. The cell pellet and supernatant fractions were
prepared as described above. Five µg of cell extract protein and
0.5 µg of supernatant protein were assayed by zymography. Urine was
collected directly (void urine) or by bladder puncture of anesthetized
mice and 1-µl volumes analyzed by zymography. Plasma was prepared
from citrated blood and 1-µl volumes were analyzed by zymography.
uPA Immunoblot and Esterase Activity Assays
Void
and bladder urine was prepared as described above and aliquots were
snap frozen in liquid nitrogen (esterase assays) and placed in sample
buffer containing 2% SDS (immunoblot assays). For immunoblot analyses,
2.5 µl of urine was fractionated by SDS-polyacrylamide gel (12%)
electrophoresis, and the proteins transferred to Immobilon P
(Millipore) membranes. Mouse uPA was detected using affinity-purified
rabbit anti-mouse uPA antibodies (56) and alkaline
phosphatase-conjugated goat anti-rabbit IgG (Sigma) and the
5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma)
staining system. The uPA esterase activity assays were performed using
the uPA-specific chromogenic substrate, Spectrozyme-UK (American
Diagnostica). Urine samples (2-5 µl) were diluted in 100
µl of 50 mM Tris-HCl buffer (pH 8.8) containing 0.01%
Tween 80, 10 kallikrein inhibitor units of aprotinin (Calbiochem), and
2.5 mM Spectrozyme-UK and incubated at 37 °C. Samples were
read at 405 nm with the rate of substrate conversion still in the
linear range.
Generation of uPAR-deficient Mice
To disrupt the
uPAR gene in mice a replacement-type targeting vector was constructed
from the cloned murine uPAR gene (Fig. 1). The vector was
designed to eliminate the third exon of the uPAR gene which encodes the
second half of the amino-terminal uPA-binding domain. Exon 3 and
neighboring intron sequences were replaced by a 6-kb selectable marker
gene encoding human HPRT (45) that was oriented in the same
direction as the uPAR gene relative to transcription. A herpes simplex
virus-thymidine kinase minigene was also incorporated to provide the
means to select against random integration(44) . The vector was
electroporated into the HPRT-deficient embryonic stem cell line E14TG2a
(46). Eleven of 86 double-resistant clones (13%) picked for DNA
analysis appeared to have incorporated the uPAR targeting vector by
homologous recombination when initially screened by PCR (data not
shown). Two positive clones were examined by Southern blot
hybridization analysis using the probes indicated in Fig. 1,
confirming the disruption of the uPAR gene (data not shown). Each of
these clonal isolates was microinjected into 3.5-day-old C57BL/6
blastocysts to generate chimeric founder mice. Four of 20 chimeric
males transmitted the targeted allele to their offspring. Crosses
between heterozygous mice resulted in progeny that were homozygous for
the targeted allele based on both Southern blot hybridization (for
example, see Fig. 2A) and PCR (data not shown).
Figure 1:
Strategy for targeting the uPAR gene by
homologous recombination. The structure of the uPAR targeting vector (A), normal mouse uPAR gene (43) (B), and targeted
uPAR gene (C). Solid boxes indicate the position of
exons (numbered 1 through 7). The 2-kb herpes simplex virus-thymidine
kinase (HSV-tk) (solid-framed) and 6-kb HPRT (cross-hatched) cassettes were introduced in the same
orientation as the uPAR gene relative to the direction of
transcription. A 1274-bp BamHI fragment containing exon 3 was
removed and replaced with the HPRT minigene in the targeted uPAR gene.
The relative position of Southern blot hybridization probes and PCR
oligonucleotide primers used for the initial screening of embryonic
stem cell clones are indicated at the bottom. The double
arrows indicate the expected fragments obtained for the normal and
correctly targeted uPAR allele in Southern blot hybridization using the
probes and restriction enzymes indicated. Bg, BglII; B, BamHI; H, HindIII; P, PvuII; and E, EcoRI.
Figure 2:
Southern blot and mutant allele transcript
analysis. A, representative Southern blot hybridization data
with tail biopsy DNA prepared from wild-type (+/+),
heterozygous uPAR (+/-), and
homozygous uPAR
(-/-) mice
digested with BglII and hybridized with the exon 2-specific
probe indicated in Fig. 1 (also see ``Experimental
Procedures''). Based on the complete nucleotide sequences of the
mouse uPAR gene (43) and human HPRT minigene (45) the expected size of
the hybridizing BglII fragments from the normal- and
targeted-uPAR alleles are 7108 and 4590 bp, respectively. B,
Northern blot hybridization analysis of total RNA (20 µg) isolated
from uPAR
(lanes 1-4),
uPAR
(lanes 5-8), and
uPAR
(lanes 9-12) embryonic
fibroblasts (second-passage). The cells were cultured with 160 nM PMA for either 25 h (lanes 1, 5, and 9) or 6.5 h (lanes 2, 6, and 10), treated with the solvent
Me
SO (10 µl/10-ml culture) for 6.5 h (lanes 3, 7, and 11), or left untreated (lanes 4, 8, and 12). The doublet observed in normal uPAR mRNA results
primarily from the use of alternative polyadenylation sites (50). The
relative positions of the 28 S and 18 S ribosomal RNAs are indicated at
the left. Ethidium bromide staining of the ribosomal RNAs
prior to blotting indicated similar loading in all lanes. C,
reverse transcriptase-PCR products generated using primers
complementary to exons 2 and 5 (see ``Experimental
Procedures'') and poly(A)
RNA template extracted
from lung (lanes 2-4), kidney (lanes 5 and 6), and upper stomach (lanes 7 and 8) of
wild-type (lane 2), uPAR
heterozygous (lanes 3, 5, and 7), and homozygous
uPAR
(lanes 4, 6, and 8) mice. The negative control reaction mixture (lane 1)
contained no RNA template and the positive control reaction mixture (lane 9) contained poly(A)
RNA extracted from
mouse skin treated with PMA for 72 h. The expected size of the PCR
product from uPAR mRNA following the normal splicing pattern is 426 bp.
The expected size of a PCR product from mutant uPAR mRNA following an
exon 2-to-4 splicing pattern (i.e. lacking the 144-bp exon 3)
is 282 bp. The relative positions and size (in bp) of molecular weight
markers are shown at the left.
Homozygous, uPAR-deficient (uPAR) mice
were indistinguishable from control littermates in appearance and
behavior. There was no indication of loss of fetal
uPAR
mice based on the Mendelian pattern of
transgene transmission; of 305 progeny raised from heterozygous
(uPAR
) parents, 76 (24.9%) were homozygous for
the wild-type allele (uPAR
), 154 (50.5%) were
heterozygous (uPAR
), and 75 (24.6%) were
homozygous for the targeted allele (uPAR
).
Likewise, crosses between uPAR
and
uPAR
mice resulted in 67 progeny of which 33
(49.3%) were uPAR
and 34 (50.7%) were
uPAR
. Furthermore, uPAR
mice were comparable to control mice in overall survival; only 2
of 63 uPAR
mice identified at weaning and
subsequently monitored for at least 4 months died spontaneously, and
the oldest uPAR
mouse in our colony has been
maintained for over 8 months.
males was 7.2
pups, a value nearly identical to the average (7.3 pups/litter) seen in
46 litters generated from heterozygous mice. Likewise,
uPAR
females had an average litter size of
8.2 pups (9 litters) when bred with uPAR
males. Finally, 15 litters were produced from crosses between
uPAR
mice with an average litter size of 7.1
pups.
Abnormal uPAR mRNA Generated from the Mutant
Allele
Earlier studies have shown that uPAR mRNA is present at
only very low levels in mouse tissues in the absence of stimulatory
factors such as injury or exposure to tumor promoting phorbol
esters(11, 50) . Therefore, to determine whether any
uPAR mRNA could be generated from the targeted uPAR allele, we prepared
cultures of embryonic fibroblasts from 17-day-old embryos collected
from a wild-type breeding pair (uPAR fibroblasts), a uPAR
breeding pair
(uPAR
fibroblasts), and a wild-type female
bred with a uPAR
male
(uPAR
fibroblasts). Total RNA extracts were
prepared from both untreated fibroblasts and cells exposed to 160
nM PMA for 6.5-25 h and then analyzed for uPAR mRNA by
Northern blot hybridization using a mouse uPAR cDNA probe. uPAR mRNA
was readily detected in Northern blot analysis of total RNA
preparations from untreated and PMA-stimulated uPAR
and uPAR
fibroblasts (Fig. 2B). Surprisingly, a uPAR transcript with reduced
intensity was also detected in total RNA extracts from PMA-stimulated
uPAR
cells. This transcript had a slightly
increased mobility, compatible with the notion that it was generated by
the splicing of exons 2 to 4 with the excision of the HPRT-cassette. To
test if this transcript was generated in this way and present in
primary mouse tissue, reverse transcriptase-PCR was performed using RNA
templates extracted from lung, kidney, and stomach of
uPAR
mice and specific uPAR primers
complementary to exon 2 and exon 5 (see ``Experimental
Procedures''). A single PCR product of the expected size (426 bp)
was generated using RNA from uPAR
and
uPAR
mice (Fig. 2C). However, a
fainter product of approximately 280 bp was observed using RNA from
uPAR
mice (Fig. 2C). This
product was essentially identical to the size expected if exon 2 were
spliced to exon 4 in mutant uPAR transcripts (i.e. 282 bp).
Thus, despite the presence of several exon-intron junctions and a
polyadenylation signal in the inserted HPRT-cassette(45) ,
stable uPAR transcripts appear to be generated from the targeted uPAR
allele where the HPRT sequences are efficiently removed to give rise to
an mRNA lacking exon 3. Nevertheless, no uPAR protein product has been
detected in any tissue of uPAR
mice (see
below and ``Discussion'').
Loss of uPAR Protein
The presence of uPAR protein
in tissues collected from uPAR and control
mice was explored using both immunological and functional assays. In
the immunological analyses, detergent-phase proteins (51, 57) were extracted from the spleen of
uPAR
, uPAR
, and
uPAR
mice and fractionated by
SDS-polyacrylamide gel electrophoresis. uPAR was then detected using
rabbit antisera raised against synthetic peptides corresponding to the
first or second domains of mouse uPAR(51, 57) .
Immunoreactive material with the expected size (i.e. 55-70 kDa; size heterogeneity of uPAR is related to
glycosylation; Refs. 51 and 57) was readily detected in extracts from
uPAR
mice using antisera specific for either
the first domain (Fig. 3A, upper left panel) or second
domain (Fig. 3A, lower left panel) of mouse uPAR.
Immunoreactivity was also detectable in extracts from
uPAR
mice but with reduced intensity (Fig. 3A, upper and lower left panels). The
55-70 kDa bands were not seen when the uPAR peptides used for
immunization were present at 10 µM concentration in
antibody incubation mixtures, indicating the specificity of these uPAR
immunoassays (Fig. 3A, upper and lower right
panels). Importantly, no specific immunoreactivity was observed
with extracts from uPAR
mice using either of
the available uPAR peptide antibodies (Fig. 3A, upper and lower left panels). Similar results were obtained in
parallel assays of uPAR antigen in lung extracts (data not shown).
Therefore, within the sensitivity of these immunoblot assays,
uPAR
mice do not appear to accumulate a
mutant uPAR polypetide lacking exon three-encoded sequences.
Figure 3:
uPAR immunoblot and ligand cross-linking
analyses. A, Western blot immunoanalysis of uPAR in spleen
extracts (lanes 3-5) from uPAR (lane 3), uPAR
(lane
4), and uPAR
(lane 5) mice
using a rabbit antiserum raised against mouse uPAR domain-1 (upper
panels) and domain-2 (lower panels) peptides. The assay
specificity was established by parallel assays in which the peptides
used for immunization were either omitted (left panels) or
included (right panels) at 10 µM in antiserum
reaction mixtures. Extracts from P388D.1 (lane 1) and Lewis
lung (lane 2) mouse tumor cell lines were included on each
blot as positive controls (51, 57). The upper bands in these control
extracts are known to be heavily glycosylated variants of uPAR (51,
57). The lower band observed in P388D.1 cell extracts using both
domain-1 and domain-2 antisera is a deglycosylated form of uPAR (51).
The lower band observed in Lewis lung carcinoma cell extracts using the
domain-2 antiserum is a cleaved form of the receptor which lacks
domain-1 (57). B, cross-linking of
I-labeled
human ATF to extracts from spleen (upper panel) and lung (lower panel) of uPAR
(lanes 3 and 4), uPAR
(lanes 5 and 6), and uPAR
mice (lanes 7 and 8). The control extracts were from
P388D.1 (upper panel) and Lewis lung tumor cells (lower
panel). In competition experiments, extracts were preincubated
with 100 nM unlabeled mouse ATF prior to addition of
radiolabeled ATF (lanes 2, 4, 6, and 8). The relative
positions of molecular weight markers (kDa) are indicated at the left.
To
assay for uPA binding activity, we took advantage of the ability to
cross-link I-labeled amino-terminal fragment (ATF) of
human uPA to mouse uPAR using
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and
sulfo-N-hydroxysuccinimide as cross-linking agents.
A radiolabeled cross-linking product having a relative mobility
on SDS-polyacrylamide gels matching the combined size of the ATF ligand
and uPAR was observed in reaction mixtures prepared with
detergent-phase proteins from the spleen, lung, and kidney of
uPAR
and uPAR
mice (Fig. 3B, and data not shown). This product was specific
as it was readily competed by addition of a 40-fold excess of unlabeled
ATF (Fig. 3B). Notably, while apparently the same
cross-linking product could be detected in extracts from
uPAR
and uPAR
mice,
only about half as much product was observed in the latter. Consistent
with the our earlier immunological findings, no specific uPAR
cross-linking product was generated using extracts from spleen, lung,
and kidney of uPAR
mice (Fig. 3B, and data not shown). In summary, despite the
generation of an abnormal uPAR mRNA lacking the third exon in
uPAR
mice, uPA binding activity was abolished
and any uPAR antigen was present at levels below the limit of detection
of our assays.
uPAR Deficiency Ablates Cell-surface Plasminogen
Activation by Peritoneal Macrophages
To directly test the
hypothesis that uPAR is important for efficient plasminogen activation
at the cell surface, thioglycollate-stimulated peritoneal macrophages
were collected from uPAR,
uPAR
, and uPAR
mice
and placed in culture. The cells were incubated with purified mouse
pro-uPA, washed, combined with plasminogen and the formation of plasmin
was followed using a chromogenic plasmin substrate(54) . A
strong stimulation of plasmin formation was apparent with macrophages
from uPAR
cells relative to control incubation
mixtures (Fig. 4). As would be expected with cells carrying half
the number of uPA receptor molecules, the rate of plasmin formation was
reduced about 50% in uPAR
macrophage cultures
relative to uPAR
cultures. Most importantly,
cells from uPAR
mice did not stimulate
plasmin formation above the background activity observed with no cells
present. These data show definitively that, at least for macrophages,
uPAR is critical in pro-uPA-mediated plasminogen activation at the cell
surface.
Figure 4:
Pro-uPA mediated cell-surface plasminogen
activation is abolished on uPAR macrophages.
Plasmin generation initiated by pro-uPA is shown as the increase in
absorbance at 405 nm for uPAR
(solid
squares), uPAR
(inverted
triangles), and uPAR
(triangles) peritoneal macrophages and control wells
blocked with 10% fetal calf serum (open squares). The data
shown are the means obtained with cells from 3 mice with determinations
made in quadruplicate. The inset shows maximal rates of
plasmin generation (±S.E.) calculated from plots of A
versust
and expressed in arbitrary units.
Cell-free uPA Accumulates in
uPAR
Since uPAR both
anchors uPA to the cell surface and ultimately mediates uPA
internalization, one expected consequence of uPAR deficiency would be
the redistribution of uPA in tissues. To test this, peritoneal lavages
collected from thioglycollate-treated mice were separated by low speed
centrifugation into a cellular (primarily macrophage) fraction and a
cell-free supernatant. The uPA in each fraction was detected by gel
zymography using equivalent quantities of total protein. The levels of
uPA in the cell-free peritoneal fraction appeared to be significantly
higher in uPARMice
mice relative to control
wild-type, tPA-deficient, and plasminogen-deficient mice (Fig. 5). In contrast, there was no apparent difference in
cell-free tPA in uPAR
mice relative to
control wild-type, plasminogen-deficient, and uPA-deficient mice, which
provides an independent internal control for sample loading (Fig. 5A). Only a modest corresponding decrease in the
amount of cell-associated uPA was observed. This small reduction is
probably a reflection of the much larger size of the intracellular uPA
pool over the cell surface-associated uPA pool in activated
macrophages, which are known to produce copious amounts of
uPA(58) . A similar uPA distribution analysis was done using
pulmonary lavage from four uPAR
and four
control uPAR
mice (Fig. 5B). A
clear decrease was observed in the amount of cell-associated
(presumably resident macrophage-associated) uPA in
uPAR
mice relative to uPAR
animals. However, in this case no corresponding accumulation of
cell-free uPA was obvious. While several explanations are possible for
this latter finding, it is likely to simply reflect the >8 times
larger size of the cell-free over cell-associated uPA pool in lung
lavage fluid collected from normal mice (data not shown).
Interestingly, no detectable differences were found in plasma uPA
levels between uPAR
,
uPAR
, and uPAR
mice
(data not shown). Likewise, no major differences were found in either
high- or low-molecular weight uPA levels in urine samples collected
from mice of all three genotypes (see below). Thus, uPAR deficiency
appears to result in a significant change in the levels or distribution
of uPA in some, but not all, tissues.
Figure 5:
Changes in uPA distribution in
uPAR-deficient mice. A, plasminogen activator zymography of
the cell-associated (lanes 1-7) and cell-free
supernatant (lanes 8-14) fractions of peritoneal lavage
fluid collected from wild-type (lanes 3 and 10),
uPAR (lanes 6, 7, 13, and 14), uPA
(lanes 4 and 11), tPA
(lanes 5 and 12), and plasminogen (Plg)-deficient (lanes 1, 2,
8, and 9) mice. Note the increased level of uPA in the
cell-free fraction from uPAR
mice (lanes
13 and 14) relative to mice with other genotypes (with no
difference in tPA). B, plasminogen activator zymography of the
cell-associated (lanes 1-9) and cell-free (lanes
10-19) fractions of pulmonary lavage fluid collected from
uPA
(lanes 1 and 10), uPAR
(lanes 2-5 and 11-15), and uPAR
(lanes 6-9 and 16-19) mice. Note
the reduced level of uPA in the cell-associated material from
uPAR
mice (lanes 6-9) relative
to uPAR
mice (lanes 2-5). The
relative position of uPA and tPA are indicated with arrows.
The position of molecular weight markers (kDa) is indicated to the left.
Activation of Pro-uPA Is Independent of uPAR in the
Urogenital Tract
Plasminogen activation in vitro is
accelerated by a feedback mechanism whereby plasmin converts inactive
pro-uPA to active, two-chain uPA. This reaction is sensitive to uPAR
with the efficiency of receptor-bound pro-uPA activation by
cell-associated plasmin being as much as 50 times higher than that
observed in solution (30). The availability of uPAR-deficient mice
provided an opportunity to study the effect of uPAR on pro-uPA
activation in vivo. Urine is a rich source of uPA, most of
which is in an active, two-chain form in normal mice(59) . To
examine the relationship between uPAR and pro-uPA activation in urine,
freshly voided and bladder urine was collected from
uPAR, uPAR
, and
uPAR
mice. The enzymatic activity and
structural form of uPA (i.e. extent of conversion to two-chain
and low molecular weight forms) in urine samples was then assayed by
esterase (chromogenic substrate) and gel zymography activity assays,
and by Western blot immunoassays. Comparable amounts of uPA activity
was found in urine samples collected from uPAR
and uPAR-expressing mice using both the esterase (data not shown)
and zymography assays (Fig. 6A). The specificity of each
of these assays was established by analysis of urine collected from
uPA
mice (Fig. 6). In addition, no
differences in the fraction of high and low molecular weight uPA
species were apparent based on zymographic analysis (Fig. 6A). Furthermore, in all cases the high molecular
weight uPA was largely in the two-chain form based on immunoblot
analysis of reduced and non-reduced samples; reduced 45-kDa uPA was
separated into 30-kDa heavy chain (Fig. 6B, right panel)
and 15-kDa light chain (eluted from gel) cleavage products. Thus, the
loss of uPAR appears to have no major impact on either the accumulation
or proteolytic activation of uPA in the urogenital tract.
Figure 6:
uPAR-independent activation of pro-uPA. A, plasminogen activator zymography of 1 µl of freshly
voided urine from uPAR (lanes 2 and 3), uPAR
(lanes 4 and 5), and uPAR
(lanes 6 and 7) mice. The specificity of the assay was controlled by
parallel analysis of 1 µl of urine from a uPA
mouse (lane 1). The position of high molecular weight (HMW) and low molecular weight (LMW) uPA is indicated
with arrows. B, immunoblot detection of uPA in 2.5
µl of non-reduced urine samples (lanes 1-6) and 2.5
µl of reduced urine samples (lanes 7-12) collected
from uPAR
(lanes 1, 2, 7, and 8), uPAR
(lanes 3, 4, 9, and 10), and uPAR
(lanes 5, 6, 11, and 12) mice. The position of high molecular weight (HMW) and low molecular weight (LMW) uPA is indicated
with arrows. The relative positions of molecular weight
markers (kDa) are indicated at the left. No immunostaining of
either the HMW or LMW protein was detected with urine from a
uPA
mouse (data not
shown).
Histologic Analysis of uPAR
A gross and microscopic survey of the major
tissues of neonatal (n = 3; 3-8 days of age),
juvenile (n = 2; 4-6 weeks of age), and adult (n = 11; 18-29 weeks of age)
uPARTissues
mice revealed no remarkable anatomical
abnormalities or pathological lesions when analyzed in parallel with
control mice. The tissues that were examined microscopically following
hematoxylin-eosin staining of formalin-fixed sections included: brain,
heart, lung, liver, kidney, adrenal, pancreas, spleen, stomach, gonads,
and intestines. Since fibrin deposition was uniformly observed in the
livers of young plasminogen-deficient mice (68) and occasionally
observed in the livers of uPA-deficient mice(60) , liver
sections of uPAR
(n = 8;
4-21 weeks of age) and control uPAR
(n = 7; 4-21 weeks of age) and wild-type (n = 1; 4 weeks of age) mice were examined
immunologically for fibrin(ogen) deposits using a specific rabbit
antiserum raised against mouse fibrinogen. Although the immunostaining
system employed was very effective in detecting hepatic fibrin deposits
in plasminogen-deficient mice(68) , only normal sinusoidal
staining of fibrinogen was found in uPAR
and
control liver sections (data not shown). A similar immunological
analysis of lung sections from uPAR
mice (n = 3; 8-12 weeks of age) also revealed no
significant fibrin deposits (data not shown).
mice. Although a thorough survey of
mouse tissues and proteases has not been completed, based on a
preliminary analysis of tissues by in situ hybridization with
uPA, tPA, plasminogen activator inhibitor-1, and several
metalloprotease-specific probes, there is no indication of any change
in the expression pattern of either related or unrelated extracellular
proteases.
(
)The absence of uPAR does alter the
distribution of the uPA ligand, at least in some tissues. Nevertheless,
there appears to be no pathology associated with either lack of cell
surface-sequestration of uPA or increase in ``free'' uPA in
either uPAR
or uPAR
mice. Any potential negative impact of released pro-uPA in
uPAR
tissues might be abrogated by factors
such as the inefficiency of plasminogen activation when uPA and
plasminogen are not cell surface-associated(30) .
mice was found to be in the
two-chain form. However, it should be noted that the increased
efficiency of receptor-bound pro-uPA activation has only been
documented with plasmin, which is itself not critical for pro-uPA
activation in the urogenital tract; the fraction of uPA in the urine
that is two-chain is virtually the same in normal and
plasminogen-deficient mice(68) . Whatever the protease that
activates pro-uPA in urine, it does not depend on uPAR for efficient
cleavage. Further studies are required to determine whether pro-uPA
activation in the urogenital system is an exception, or whether pro-uPA
activation (or initial pro-uPA activation) commonly occurs irrespective
of uPAR and/or plasmin in other tissues.
-macroglobulin, which mediates the uptake and
turnover of uPA
PA inhibitor complexes(40) . It has also
been reported that uPAR binds with high affinity to the extracellular
matrix protein, vitronectin, and this interaction is promoted by
concurrent uPA binding(41) . The combined interaction of uPAR
with uPA, vitronectin, membrane-associated proteins, and perhaps other
ligands may provide a means to simultaneously focus proteolysis, direct
cell migration, and cue locomotion-related intracellular events.
However, the results reported here show that if uPAR is truly
multifunctional, with roles outside of plasminogen activation, then
none of these roles appears to be critical to the development, overall
health, and fertility of mice that are unchallenged by disease,
infection, injury, or other functional deficits.
uPAR
mice and cells derived therefrom, should
be useful in future studies confirming the role of uPAR in binding
vitronectin and other ligands, and for exploring in more detail the
role of uPAR in uPA turnover, signal transduction, and directed cell
migration through extracellular matrices and fibrin.
mice; however, since these immunoassays
have limited sensitivity, the presence of small amounts of a
nonfunctional uPAR polypeptide in these animals has not been formally
excluded. The absence of the mutant protein may be due to protein
instability, possibly stemming from the residual odd cysteine
(Cys
encoded by exon 2 is normally linked to Cys
encoded by exon 3; Ref. 61), the loss of the carbohydrate
addition site encoded by exon 3 (Asn
; Ref. 62), and/or
aberrant protein folding.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.