PAI-1 is the major negative regulator of plasmin formation and fibrinolysis . Accumulation of PAI-1 at sites of fibrin formation inhibits fibrinolysis and matrix metallo-proteinase (MMP) activation and it has a vital role in matrix remodeling . Induced expression of PAI-1 accompanies wound repair in vitro and in vivo[15,23]. Appropriate expression of the inhibitor is necessary to prevent both premature as well as excessive proteolysis, both of which will affect normal tissue repair/remodeling processes. While elevated levels are associated with artherosclerosis and thrombosis, PAI-1 deficiency leads to increased fibrinolysis and bleeding . PAI-1 gene expression can be modulated by various factors including growth factors, hormones, endotoxins and cytokines, acting at either the transcriptional or post-transcriptional levels [25–31]. The 3.2-kb species of human PAI-1 mRNA, characterized by a long-3′-UTR, has a relatively short half-life . Growth factors and hormones regulate PAI-1 gene expression by increasing the stability of the unstable 3.2-kb PAI-1 mRNA species . Our study reports the stability of the 3.2-kb PAI-1 mRNA species caused by iron depletion in normal human lung fibroblasts.
Iron depletion was caused by exposure to deferoxamine, an iron chelator, which binds stoichiometrically to ferric iron available in free form in the intracellular pool [34,35]. It is widely used in in vitro studies on intracellular iron, to deplete the cells of transient low-molecular-weight iron pool . It is reported to be effective in increasing the expression of vascular endothelial growth tissue, GLUT-1, transferrin receptor, cyclooxygenase (COX)-2 and decreasing XOR activity [11,37–39]. Deferoxamine caused a dose-dependent increase in PAI-1 expression relative to both the controls, in all the fibroblast cell lines studied. The specificity control with iron-presaturated deferoxamine confirmed that the effect was specifically due to iron depletion, since ferric citrate combines at a 1 : 1 ratio with deferoxamine, rendering it unavailable to act on intracellular iron pool. Iron modulates the expression of several genes either at the transcriptional or post-transcriptional level. In the current study, DRB- and cycloheximide-based experiments emphasized a post-transcriptional regulation that depended on de novo protein synthesis. DRB, an inhibitor of polymerase II-mediated transcription , failed to abrogate the deferoxamine-induced steady-state level of PAI-1 mRNA. A similar post-transcriptional influence has been reported on transferrin receptor mRNA, COX-2 and XOR activity [22,41]. Ongoing protein synthesis played a crucial role in deferoxamine-mediated stability of PAI-1 mRNA. Protein synthesis inhibition causes PAI-1 mRNA accumulation or super-induction [15,42–44]. We observed similar results, where cycloheximide by itself caused PAI-1 mRNA accumulation in TIG 3-20 cells. However, deferoxamine did not induce further PAI-1 mRNA accumulation in the presence of cycloheximide. A similar inhibitory effect of cycloheximide on deferoxamine action has been previously observed in GLUT-1, COX-2 and transferrin receptor expression [9,39,45]. Deferoxamine-induced PAI-1 mRNA stability may be dependent on the synthesis of protein factor(s). Subsequent EMSA and UV crosslinking experiments revealed the presence of an 81-kDa nuclear protein, which seemingly interacted with the 3′-UTR of PAI-1 mRNA in an iron-sensitive manner. This mRNA–protein complex formation could regulate PAI-1 mRNA stability in an iron-dependent manner. Reports on protein factors binding to and regulating the stability of mRNAs are profuse . These proteins can occur in either the cytoplasm  or nucleus [48–51], in addition to shuttling between the two compartments [46,52]. The best studied example of mRNA stabilizing proteins is the 98-kDa iron responsive protein 1 (IRP1), which stabilizes transferrin receptor mRNA in an iron-responsive manner , in addition to modulating the expression of other genes such as ferritin, m-aconitase, EACA, etc. . Iron deprivation causes a structural conformation change in IRP1, increasing its affinity to the mRNAs. Another cytoplasmic protein, IRP2 (105 kDa), specifically modulates the ferritin expression in an iron-responsive fashion [41,54]. In this case, iron deprivation prevents the degradation of IRP2 which subsequently binds to and stabilizes ferritin mRNA. Since PAI-1 mRNA stability induced by iron deprivation was dependent on de novo protein synthesis, it is interesting to hypothese that iron depletion increased the expression of a protein that is expressed at low levels in normal conditions. A greater amount of protein could result in more protein–RNA complex formation and subsequent stabilization of PAI-1 mRNA. The iron-responsive proteins, IRP1 and IRP2, bind specifically to sequences termed iron-responsive elements occurring at either 3′-UTR or 5′-UTR of the mRNAs [55–57]. These consensus iron-responsive sequences are lacking in the 3′-UTR of PAI-1 mRNA, ruling out the possibilities of IRP1 or IRP2 binding to PAI-1 mRNA. We assume that a protein other than IRP1 or IRP2 interacts with PAI-1 mRNA in an iron-responsive manner. Reports on PAI-1 mRNA binding proteins are also emerging with discoveries of cytoplasmic proteins destabilizing PAI-1 mRNA in human lung carcinoma  and rat hepatoma cells . We believe that our protein is novel in that it differs from the above-mentioned proteins in size (81 kDa), location (nuclear) and function (stabilizes PAI-1 mRNA). Further work on isolating and characterizing the protein is ongoing in our laboratory.
PAI-1 has a broad array of biological activity. This study reveals the functional efficacy of the increased PAI-1 expression in suppressing cell-surface plasmin generation, an event capable of affecting cell-mediated proteolysis and migration.
In conclusion, this study demonstrates that cellular iron status regulates the expression of PAI-1 via mRNA stability and subsequently the cell-surface plasmin activity in cultured human lung fibroblasts. Low intracellular iron availability stabilizes PAI-1 mRNA, with the probable involvement of a nuclear protein that shows high specificity for the 3′-UTR of PAI-1 mRNA.