©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Receptor for Urokinase-type Plasminogen Activator Is Not Essential for Mouse Development or Fertility (*)

Thomas H. Bugge (1) (4)(§), Theodore T. Suh (1)(¶), Matthew J. Flick (1), Cynthia C. Daugherty (2), John R (4), Helene Solberg (4), Vincent Ellis (3), Keld Danø (4), Jay L. Degen (1)(**)

From the (1)Divisions of Basic Science and (2)Pathology, Children's Hospital Research Foundation, Cincinnati, Ohio 45229, the (3)Thrombosis Research Institute, Emmanuel Kaye Building, London SW3 6LR, United Kingdom, and the (4)Finsen Laboratory, Rigshospitalet, 2100 Copenhagen Ø, Denmark

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

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),()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.

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 10 to 10M) 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 uPAinhibitor complexes from the cell surface(40) .

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.


EXPERIMENTAL PROCEDURES

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% MeSO 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.


RESULTS

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 MeSO (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.

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 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 Aversust and expressed in arbitrary units.



Cell-free uPA Accumulates in uPARMice

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 uPAR 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 uPARTissues

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) uPAR 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).


DISCUSSION

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 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) .

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 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.

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/-macroglobulin, which mediates the uptake and turnover of uPAPA 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.

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 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.

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.


FOOTNOTES

*
This work was supported in part by National Grant-in-Aid 92-1103 from the American Heart Association with funds contributed in part by the Ohio affiliate (to J. L. D.), National Institutes of Health Grant HL47826 (to J. L. D.), and by funds from the Danish Cancer Society (to K. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by fellowships from the Danish Medical Research Council, the Danish Cancer Research Foundation, and the Danish Plasmid Foundation.

Supported by a fellowship from the University of Cincinnati Medical Science Scholars Program.

**
This study was done during the tenure of Established Investigatorship 93002570 from the American Heart Association. To whom correspondence should be addressed: Children's Hospital Research Foundation, TCHRF 2025, 3333 Burnet Ave., Cincinnati, OH 45229-3039. Tel.: 513-559-4679; Fax: 513-559-4317.

The abbreviations used are: uPA, urokinase-type plasminogen activator; uPAR, urokinase-type plasminogen activator receptor; tPA, tissue-type plasminogen activator; HPRT, hypoxanthine phosphoribosyl-transferase; PCR, polymerase chain reaction; ATF, amino-terminal fragment; PMA, phorbol myristate acetate; bp, base pair(s); kb, kilobase(s).

H. Solberg and K. Danø, manuscript in preparation.

J. R, unpublished results.


ACKNOWLEDGEMENTS

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.


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