The vitronectin-binding domain of plasminogen activator inhibitor-1 plays an important functional role in LPS-induced lethality in mice

Plasminogen Activator Inhibitor-1 (PAI-1) is a member of the SERine Protease INhibitor (SERPIN) superfamily and is the main physiological inhibitor of urokinase (uPA) and tissue-type plasminogen activator (tPA) [1]. Studies using PAI-1 deficient (PAI-1^−/−) mice, have demonstrated that PAI-1 not only regulates the fibrinolytic system but also modulates other physiological and pathophysiological processes, including inflammation, angiogenesis, tumor growth, and cardiovascular disease [2–6]. Regions of human PAI-1 that are critical for inhibition of plasminogen activation, binding to vitronectin (VN), and binding to Low-density lipoprotein Receptor-related Protein (LRP) have been identified [7–9]. Our laboratory has demonstrated that these functional sites are conserved in the murine system [10]. In order to define functional roles for domains within PAI-1, we generated mice that express PAI-1 with altered VN binding capacity. Binding of VN to PAI-1 stabilizes the biological activity of PAI-1 and prolongs its half-life in plasma [11–13]. Moreover, the binding of VN by PAI-1 modulates cell adhesion and cell migration via limiting VN from binding to its integrin receptor and uPAR [13–17].

It has been demonstrated that a 123 (Q→K) mutation in PAI-1 results in a significant reduction in the capacity of PAI-1 to bind to VN [18] and transgenic mice overexpressing this PAI-1 variant have been generated and characterized [19]. Previous studies have shown that a recombinant murine PAI-1 variant which has a double mutation at amino acid 101 (R→A) and 123 (Q→K) has significantly diminished capacity to bind to VN even more so than the single mutation at amino acid 123 [10]. Based on these findings, genetic mutations that translate into these alterations (R101A and Q123K) of PAI-1 were targeted into the PAI-1 gene in the mouse genome. A 2806 bp PCR genomic fragment containing PAI-1 exon 2 and exon 3 was subcloned into pCR.21-TOPO vector as the 5′ flank for the targeting vector (TV) and nucleotide substitutions were introduced by site-directed mutagenesis to generate the R101A and Q123K changes in exon 3. A 3674 bp PCR genomic fragment containing PAI-1 exons 4 and 5 was subcloned into pCR.21-TOPO vector as the 3′ flank for the TV. The 5′ flank and 3′ flank were cloned into the multicloning site of a pre-made TV backbone in which the NEO cassette was flanked by two lox P sites and two flippase recombination target (FRT) sites to yield the final TV for PAI-1 VN with R101A and Q123K mutations (Figure 1A).

Figure 1.

(A) The final targeting vector for the PAI-1^R1010A/Q123K. PAI-1 exons are black rectangles and the red circle shows the location of R101A and Q123K mutation in exon 3.The red vertical bar at the edge of the neomycin resistance gene (NEO) site is FRT site. The triangles represent Lox P site. The cytosine deaminase cassette (CDA) was inserted for negative selection and was achieved with 5-fluorocytosine. Positive selection of embryonic stem (ES) cells containing the NEO gene was made via G418 resistance. (B) WT, homologously recombined, and final alleles with arrows representing location of primers for genotyping. The gel on the left shows PCR amplicons confirming homologous recombination of the TV with the WT allele and incorporation of the Lox-FRT-NEO-FRT-LOX cassette. The right gel shows PCR amplicons of WT and final mutated allele (PAI-1R1010A/Q123K). Lox-FRT-NEO-FRT-LOX cassette was inserted between exon 3 and exon 4. This cassette was removed by breeding with transgenic mice expressing flippase. The forward primer was 5′-GCTCAACATGAGCCTAATGGATC and the reverse primer was 5′-CATTCATGAGTTCCTGGCTCCAG. These primers generate a 484 bp amplicon in WT and a 646 bp amplicon for PAI-1^R101A/Q123K. (C) Characterization of WT (White bar) and PAI-1 ^R101A/Q123K (black bar) mice 8h after injecting LPS (2 μg/g body weight) for inducing PAI-1. (a) PAI-1 antigen levels (F7 mice, n=3 for WT and n=5 for PAI-1 ^R101A/Q123K mice). (b) plasma PAI-1 inhibitory activity toward tPA relative to WT activity (F1 mice, n=3 for WT and n=4 for PAI-1 ^R101A/Q123K mice). (c) PAI-1 mRNA levels in liver tissue relative to WT (F7 mice, n=3 for WT and n=5 for PAI-1 ^R101A/Q123K mice). The detection of PAI-1 was performed using murine PAI-1 total antigen assay by ELISA. For mRNA analyses, the values indicate the fold difference relative to RPL19 and converted to fold differences relative to WT mice. Values are expressed as the mean ± S.E.M. (D) Survival of WT, PAI-1^−/−, and PAI-1 ^R101A/Q123K after LPS challenge. LPS was injected i.p. (10μg/g mouse body weight). The data are presented as Kaplan-Meier survival curves. WT; n=33, PAI-1; n=25 and PAI-1 ^R101A/Q123K n=36 (F7). Survival rate differences were compared using the log-rank test. P values: WT vs PAI-1^−/− =0.03, WT vs PAI-1 ^R101A/Q123K =0.011.

The TV was electroporated into C57BL/6/129 embryonic stem (ES) cells. The ES cells surviving with negative selection with 5′-fluorocytosine for cytosine deaminase cassette (CDA ) gene and positive selection with G418 for the neomycin resistance (neo) gene, were screened by southern blot analysis and the mutations confirmed by PCR (data not shown). A PCR strategy was also employed to confirm homologous recombination (Figure 1B). Recombined ES cells were injected into blastocysts and chimeric males were identified. The resulting F1 offspring from chimeric male mice crossings with C57BL/6 female mice were tested for proper germline transmission by PCR and sequence analysis (Figure 1B). F1 mice were then bred with transgenic mice expressing flippase, Tg-CAG_FLPe37, to remove the neo gene (Figure 1B). The PCR forward primer: 5′-GCTCAACATGAGCCTAATGGATC and reverse primer: 5′-CATTCATGAGTTCCTGGCTCCAG were used to detect the removal of the neo gene. A PAI-1 genomic fragment from PAI-1 ^R101A/Q123K mice was cloned and sequenced and it was found to contain the mutations for R101A, Q123K, and the FLPe/FRT recombination sequence. Blood counts, blood analyses, body weights, and litter sizes were determined for WT, PAI-1^−/−, and PAI-1^R101A/Q123K mice and all were within the normal range.

Lipopolysaccharide (LPS) is derived from the outer membrane of gram-negative bacteria and is a known inducer of PAI-1 gene expression [20]. In order to determine if this response is equivalent between WT and PAI-1^R101A/Q123K mice, LPS (2 μg/g body weight, E.C. 0111:B4, Sigma) was injected, i.p., into 8–12 week male WT and PAI-1^R101A/Q123K mice. After 8 hr, plasma PAI-1 levels and PAI-1 inhibitory activity were determined. Plasma levels of PAI-1 in WT and PAI-1^R101A/Q123K mice were equivalent (Figure 1Ca), as well as plasma PAI-1 inhibitory activity (Figure 1Cb). In addition, the mRNA levels of PAI-1 in liver were also the same between the two genotypes (Figure 1Cc). These results indicated that PAI-1^R101A/Q123K mice have the same ability to produce PAI-1 and the PAI-1 from PAI-1 ^R101A/Q123K mice maintained plasminogen activator inhibitory activity. LPS is the major causative agent in gram-negative endotoxemia. Hallmark features of this disease are systemic inflammatory response and a hypercoagulablility. The systemic microthrombosis that develops leads to disseminated intravascular coagulation (DIC) and subsequently hypoxic organ failure [21–23]. Indeed, our laboratory has previously shown that a coagulation factor VII deficiency protects against lethal endotoxemia [24]. Thus a balance between the fibrinolytic system and the coagulation system plays an important role in regulating downstream effects of endotoxemia. Since PAI-1 is a target following LPS exposure, it was determined whether a lack or mutation of PAI-1 would affect survival after exposure to a lethal dose of LPS. For this study, 8–12 week male WT, PAI-1^−/−, and PAI-1^R101A/Q123K mice were injected, i.p., with LPS (10 μg/g body weight) and survival monitored every 3 hr. PAI-1^−/− and PAI-1^R101A/Q123K mice were shown have significant (P=0.03, P=0.011, respectively) survival advantage relative to WT (Figure 1D). Studies have demonstrated that vitronectin levels in lungs following intratracheal administration of LPS are significantly increased [25]. Utilizing this model, vitronectin null mice were protected against LPS-induced acute lung injury [25]. Additionally, other studies have demonstrated that there is an increased occurrence of neutrophil apoptosis in LPS-treated vitronectin null mice [26] apoptotic cells can protect mice from LPS toxicity [27]. In the current study PAI-1 plays a critical role in LPS-induced lethality and the VN binding capacity of PAI-1 is important for this function potentially through the enhanced effect of VN on PAI-1 inhibition of the anti-coagulant, anti-inflammatory protein, Protein C [28].