No abstract

Full-length cDNA for plasminogen activator inhibitor (PAI-1) was isolated from a human umbilical vein endothelial cell (HU-VEC) lambda gtl1 cDNA library. Three overlapping clones were identified by immunologic screening of 10' recombinant phage using a rabbit anti-human fibrosarcoma PAI-1 antiserum. The fusion proteins encoded by these three clones also react strongly with a monoclonal mouse anti-human fibrosarcoma PAI-1 antibody. By nucleotide sequence analysis, PAI-1 cDNA encodes a protein containing 402 amino acids with a predicted, nonglycosylated molecular mass of 45 kD. Identity of this material as authentic PAI-1 was confirmed by the presence of high level homology with the primary amino acid sequence of an internal peptide prepared from purified rat hepatoma PAI-1. The predicted amino acid sequence also reveals extensive homology with other members of the serine protease inhibitor gene family. Cultured HUVECs contain two PAI-I mRNA species, both encoded by a single gene, differing by 1 kb in the 3' untranslated region. The PAI-i gene is located on human chromosome 7.

The secretions of interleukin 1 (IL-1), tumour necrosis factor alpha (TNF), and prostaglandin E2 (PGE2) of low-dose E. coli lipopolysaccharide (LPS)-stimulated human monocytes (M phi) were investigated in an endotoxin (ET)-free milieu (less than 1.6 pg LPS/ml). Human M phi cultures from nine healthy men were stimulated with 0, 12.5-500, and 250,000 pg LPS/ml as measured by a very sensitive Limulus test. The IL-1 activity was tested by the mouse costimulatory thymocyte (LAF) assay, which was thoroughly standardized and characterized (interassay variation 22-24%, intra-assay variation 3-7%). Spontaneous M phi secretions of IL-1, TNF, and PGE2 were negligible, but 12.5 pg LPS/ml significantly stimulated the secretions of these M phi products and the monokine responses to 500 and 250,000 pg LPS/ml were almost in the same range. It was demonstrated that the secretions of IL-1-TNF and TNF-PGE2 were strongly correlated. Pronounced interindividual differences in LPS responsiveness were demonstrated, and two low-responders, one of whom was HLA-DR1,2-positive, were identified. Three first-degree relatives of the DR1,2-positive low-responder had similar low responses. Furthermore, M phi cultures were prepared weekly for 4 weeks from four HLA-DR different men and the only DR2,2 homozygous individual had low monokine responses. In conclusion, stable interindividual differences in in vitro monokine and PGE2 secretions of LPS-stimulated M phi were demonstrated. It is suggested that HLA-DR2-positive individuals may be low responders.

Abstract. We studied the immunocytochemical localization of urokinase-type plasminogen activator (u-PA) and the type 1 plasminogen activator inhibitor (PAI-1) in human fibroblasts and sarcoma cells, using both polyclonal and monoclonal antibodies. The u-PA was found to be located at discrete cell-substratum contact sites, and also at areas of cell-cell contacts, whereas PAI-1 was distributed as a homogenous carpet excluding strialike areas on the substrate under the cells. To confirm the extracellular localization of u-PA and PAIl, we stained the cells live at 0°C before fixation. A double-labeling experiment showed different distribution of u-PA and PAI-1 under the cells, and especially their peripheral parts. The staining pattern of u-PA and PAI-1 resisted treatment with 0.2% saponin followed by mechanical removal of cells, a method previously reported to isolate focal contact membranes of fibroblasts. We further demonstrated the deposition of u-PA to the contact areas of cells obtained by saponin treatment by zymography, and that of PAI-1 by metabolic labeling, reverse zymography, immunoblotting, and immunoprecipitation. Fibronectin was also present in the preparations. The deposition of both PAI-1 and fibronectin by the sarcoma cells was enhanced, after treating the cells with 10 -6 M dexamethasone. The confinement of u-PA to discrete contact sites and the more uniform distribution of PAI-1 on the cell substratum may explain how cells producing large amounts of enzyme inhibitors can produce PAmediated focal proteolysis.

Abstract.We have recently shown that urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitor type 1 are both found extracellularly beneath cultured human skin fibroblasts and HT-1080 sarcoma cells, but in distinct localizations. Here, the ultrastructural distribution of u-PA was studied using immunoferritin electron microscopy. In HT-1080 cells, u-PA on the extracellular aspect of the plasma membrane was detected at sites of direct contact of the cell with the growth substratum beneath all parts of the ventral cell surface. The ferritin-labeled adhesion plaques, which were enriched in submembraneous microfilaments, were frequently seen at the leading lamellae of the cells as well as in lamellipodia and microspikes. Besides the cell-substratum adhesion plaques, ferritin label was detected at cell-cell contact sites. Double-label immunofluorescence showed a striking colocalization of u-PA and vinculin in both HT-1080 cells and WI-38 lung fibroblasts, which is consistent with u-PA being a focal contact component. The u-PAcontaining focal contacts of WI-38 cells had no direct codistribution with fibronectin fibrils. In WI-38 cells made stationary by cultivation in a medium containing 0.5 % FCS, vinculin plaques became highly elongated and more centrally located, whereas u-PA immunolabel disappeared from such focal adhesions. These findings show that plasma membrane-associated u-PA is an intrinsic component of focal contacts, where, we propose, it enables directional proteolysis for cell migration and invasion. rIESIVE interactions of cells with the extracellular matrix and their reversal are critical events for morphogenetic movements during embryonic development and for cancer cell invasion during metastasis (30, 49). The molecular structures at the sites where cultured cells contact each other and the growth substratum are thus of great interest. Focal contacts, the sites of closest cell-substratum apposition, can be visualized either by electron microscopy (1) or by interference reflection microscopy (23). Focal contacts are localized at the termini of actin-containing microfilament bundles, and contain vinculin (7, 19) and talin (6) on the cytoplasmic side, but apparently do not have fibronectin on the extracellular side (9). The so-called close contacts exhibit a greater substrate separation distance, do not contain vinculin, and probably represent a more labile type of contact (23). A third kind of substrate adhesion site termed the fibronexus (42) or extracellular matrix contact site (9) is characteristic of well-spread and stationary fibroblasts. It features a codistribution of fibronectin, its plasma membrane receptor, actin, vinculin, and alpha-actinin throughout the entire contact site and is located at central regions of the cell rather than at its margins (11,15). Fibronexuses show very close transmembrane associations of actin microfilaments and fibronectin fibrils (42,43).