Patricia G. Murphy

Modulation of plasminogen activator and plasminogen activator inhibitor expression in the human U373 glioblastoma/astrocytoma cell line by inflammatory mediators

The human U373 glioblastoma/astrocytoma cell line was found to constitutively produce and secrete a plasminogen activator and a plasminogen activator inhibitor. The plasminogen activator was identified as urokinase based on apparent molecular weight, immunoblotting with anti-urokinase antibodies, and Northern blotting with a human urokinase cDNA probe. The inhibitor secreted by U373 cells was found to be related to the PAI-1 molecule based on reactivity with anti-human PAI-1 antibodies, apparent molecular weight, and Northern blot analysis with a human PAI-1 cDNA probe. The expression of both urokinase and the PAI-1-like molecule by U373 cells could be modulated by phorbol myristate acetate or by inflammatory mediators such as interferon-gamma and interleukin-1. In the case of interleukin-1, the alpha form exhibited no detectable effect while the beta form not only elevated inhibitor levels, it also appeared to induce the production of tissue plasminogen activator. Thus, in these cells interleukin-1 beta induces alterations in PA and PAI expression and interleukin-1 alpha does not, even though the two forms are reported to utilize the same cellular receptor.

Plasminogen activators and plasminogen activator inhibitors in connective tissues and connective tissue cells: influence of the neuropeptide substance P on expression

Tissue segments isolated from ligament, epiligament, and synovial tissues from mature female New Zealand White Rabbits were demonstrated to constitutively secrete a plasminogen activator. Several tissues were also observed to constitutively secrete a plasminogen activator inhibitor which was detected in the form of a PA-PAI complex. Heterogeneity was observed in PA and PAI activity between the different connective tissues. Heterogeneity also existed between and within the medial collateral (MCL), lateral collateral (LCL), and the anterior cruciate (ACL) ligaments. In addition to the differences in constitutive expression of PA and PAI activity, differences in the responsiveness to the neuropeptide substance P (10(-5)-10(-9) M) were also detected. This responsiveness to substance P was displayed by an increase in PA and PAI activity in the conditioned medium. The pattern of responsiveness reflected the degree of innervation of these tissues. That is, synovium and epiligament tissue were the most responsive tissues to substance P while the MCL, LCL and ACL were less responsive to the neuropeptide. Parallel results were obtained using cell culture with fibroblasts isolated from the above mentioned tissues. That is, the pattern of responsiveness was similar between cells and tissue segments. More specifically, cells isolated from both synovium and epiligament increased their both their PA (slightly) and PAI activity following exposure to substance P. This was demonstrated at both the protein and RNA level. Thus, cells within a tissue maintain their phenotype when removed from their three-dimensional matrix. These results are unique in demonstrating that normal ligament and synovial cells and tissue respond to substance P by altering the expression of PA and PAI activity. This investigation further supports the concept that innervation may be important in normal connective tissue function.

Human HuH-7 hepatoma cells express urokinase and plasminogen activator inhibitor-1: identification, characterization and regulation by inflammatory mediators.

The human hepatoma HuH-7 cell line was shown to constitutively express both a plasminogen activator (PA) and a plasminogen activator inhibitor (PAI). Four sublines of the HuH-7 cell line were analyzed and found to express differing amounts of both PA and PAI. The plasminogen activator produced by these cells was identified as urokinase based upon molecular weight, inhibition of activity with anti-UK but not anti-t-PA antibodies, adherence to an anti-UK affinity column and by Northern blotting demonstrating positive hybridization with the cDNA for UK, but not with the t-PA cDNA. The inhibitor produced by HuH-7 cells was identified as PAI-1 by molecular weight, immunoblotting techniques, adherence to an anti-PAI-1 affinity column, and by Northern blotting demonstrating positive hybridization with the cDNA for PAI-1, but not with the PAI-2 cDNA. The expression of both UK and PAI-1 by HuH-7 cells could be modulated by cytokines known to influence the acute phase response. The addition of interleukin-1 (IL-1) induced the expression of both UK and PAI-1. The increase of PAI-1 was due to an increase in amount of the PAI-1 mRNA. The presence of both interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF) also increased UK and PAI-1 levels, although not as dramatically as IL-1. The addition of IL-1 together with IL-6 produced a slight synergistic response with respect to PAI-1 expression. This suggests that PAI-1 is able to respond to mediators which aid in the induction of the acute phase response. These studies demonstrate that cells of liver origin are able to produce components of the fibrinolytic system. The synthesis of these components can be altered by inflammatory mediators and thus may be involved in hepatic regulation of fibrinolysis in both normal and diseased states.

Expression of plasminogen activator and plasminogen activator inhibitor by rat mesothelioma induced by asbestos

We have investigated the expression of plasminogen activators (PAs) and PA inhibitors (PAIs) by an asbestos-induced mesothelioma. Using zymographic, immunological and biochemical techniques it was demonstrated that cell lines derived from the tumor express high levels of PAI and low levels of a UK-like PA. Adherent and partially non-adherent variants of the mesothelioma expressed indistinguishable amounts of PAI and UK. Based on partial biochemical characterization, the PAI secreted by the mesothelioma cells was a set of PAIs which consisted of PAI-1 in addition to other species. These observations indicate that the difference in growth phenotype of the adherent and partially non-adherent lines was not due to detectable differences in PA and PAI expression.