Monocytes, and their more differentiated counterparts macrophages, represent a population of haematopoietic cells which play a vital role in orchestrating and effecting immune responses. One of the products of activated monocytes and macrophages is the serpin, plasminogen activator inhibitor type 2 (PAI-2), a specific inhibitor of the serine proteinase, urokinase-type plasminogen activator (uPA). In the present study, the regulation of PAI-2 expression in monocytic cells in response to agents inducing monocyte activation was examined. The contribution of PAI-2, as an extracellular inhibitor of fibrinolysis mediated by uPA, was also assessed in relation to the fibrinolytic and coagulative capabilities of monocytic cells. In addition the novel finding that PAI -2 may have a second function as a modulator of cell death processes was investigated.
PAI-2 synthesis is associated with tumours of myelomonocytic origin, as demonstrated both in vivo and in studies employing myelomonocytic leukaemic cell lines. Several myeloid leukaemic cell lines representing cells arrested at different stages of myelomonocyte differentiation, were examined for expression of PAI-2 in response to monocyte activating agents by Northern and Western blot analysis. It was demonstrated that PAI-2 mRNA and protein expression were associated with monocyte activation and differentiation toward the macrophage cell lineage induced by agents such as the phorbol ester PMA, TNF and combinations of cytokines. LPS and poly I/C, which simulate exposure to bacterial and viral infection respectively, also induce rapid induction of PAI-2 gene expression in monocytic cells. Taken together, these results suggest that PAI-2 is synthesised as part of the early response of monocytes to infection and agents inducing activation of these cells.
Activated monocytes are capable of synthesis of several mediators of fibrinolysis and coagulation in response to activating agents. PAI-2 expression was therefore investigated in relation to tissue factor, which promotes coagulation, and the anti-fibrinolytic mediator, plasminogen activator inhibitor type 1 (PAI-1). It was demonstrated that PAI-2 is co-expressed with tissue factor in differentiated monocytic cells, whilst PAI-1 was not found to be a major product of these cells. Thus while the synthesis of tissue factor by activated monocytes promotes fibrin deposition, co-expression of PAI-2 may prevent dissolution of fibrin deposits and this may contribute to the coagulopathies associated with monocytic leukaemia.
PAI-2 synthesis in response to activating agents was demonstrated to be both rapid and transient in myeloid leukemic cells. The mechanisms responsible for this induction were investigated using the promyelocytic cell line, HL60. Run-on transcription analysis demonstrated that the initial rapid induction of PAI-2 in response to the differentiating agent, PMA, was predominantly due to increased gene transcription. Inhibition of protein synthesis by cycloheximide resulted in stabilisation of the PAI-2 mRNA transcript, observed both in resting and PMA stimulated cells, suggesting that post-transcriptional regulatory mechanisms also control steady state levels of PAI-2 rnRNA. Actinomycin D-decay experiments demonstrated that the PAI-2 transcript was unstable, with a half-life of approximately 30 minutes. Inhibition of protein synthesis by cycloheximide stabilised the PAI-2 mRNA transcript, indicating that post-transcriptional mechanisms may control PAI-2 mRNA turnover. These data demonstrate that PAI-2 gene expression is regulated both by transcriptional and post-transcriptional mechanisms. The short half-life and profile of PAI-2 mRNA expression are similar to those observed for a number of early response genes, including cytokines and oncogenes.
One mechanism by which early response genes may be regulated at a posttranscriptional level involves the regulation of the stability of mRNA transcripts, often involving AU-rich sequence determinants present in the 3'untranslated regions (3'UTRs). The role of the 3'UTR of the PAI-2 mRNA transcript in modulating mRNA stability was examined by studies in which the 3'UTR of PAI-2 mRNA was fused to the normally stable βglobin gene. Assay of the stability of the chimeric βglobin/PAI-2 3'UTR mRNA transcript by DRB-decay analysis demonstrated a decrease in the half-life of the chimeric transcript by approximately 10-fold. In order to determine the specific sequences responsible for the destabilisation of the chimeric transcript. a series of deletions within the PAI-2 3'UTR were generated using Exonuclease III, and fused to βglobin. A 63 bp AU-rich destabilising element was identified, which was found to contain a 45 bp motif homologous to a destabilising determinant in the 3 'UTRs of gmCSF and c-fos. These data demonstrate that the 3'UTR of PAI-2 contains sequences which regulate mRNA stability and further suggests that PAI-2 gene expression may be controlled through mechanisms similar to those controlling cytokine and growth regulatory genes.
The functional role of PAI-2 in monocyte biology has thus far been considered largely from the perspective of PAI-2 as an inhibitor of uPA-mediated extracellular matrix degradation. More recently, an alternative function for PAI-2 as a protective protein in TNF-mediated cytolysis has been proposed [Kumar, S., and Bagliomi, C., (1991) Journal Biological Chemistry vol. 266: 20860-20964]. In order to investigate this alternative function, PAI-2 was stably expressed in the TNF-sensitive human cervical carcinoma cell line, HeLa. Measurement of the sensitivity of PAI-2 expressing HeLa cells to TNF mediated cytotoxicity demonstrated that expression of PAI-2 confers protection from TNF cytotoxicity in the presence of cycloheximide when compared with wild-type cells or cells transfected with antisense PAI-2 or a vector control and further that the level of PAI-2 expressed by the cells is correlated with cell survival. Morphological criteria and DNA fragmentation studies demonstrate that PAI-2 protected cells from TNF induced apoptosis. PAI-2 expression did not however protect cells from apoptosis induced by UV or ionizing radiation damage or from cell death mediated by cytotoxic T cells, natural killer or lymphocyte activated killer cells. Expression of PAI-2 was, however, protective against the cytopathic effects of selected viruses and the synergistic cytotoxic effects of TNF and IFNƴ.
Cell surface uPA did not appear to be the target of PAI-2 action as specific inhibitors of uPA and anti-uPA antibodies did not affect the sensitivity of the transfectants to TNF induced apoptosis. However, a HeLa transfectant expressing a mutant form of PAI-2, with no serine protease binding activity, was sensitive to TNF-mediated apoptosis suggesting that an intracellular proteinase other than uPA may be the target of PAI-2 protective activity.
In summary, the results reported in this study show that PAI-2 plays an intrinsic role as an early response gene in activated monocytes, and also functions to modulate selected cell death pathways. These findings may have novel implications for monocyte biology and the function of monocytes and macrophages in immune defence mechanisms.