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Fibrin Fragment Response Elements in the Plasminogen Activator Inhibitor Gene^*

^* From the Divisions of Pulmonary and Critical Care Medicine and Pediatric Pulmonology, University of Alabama at Birmingham, Birmingham, AL.

Correspondence to: M.A. Olman, MD, Division of Pulmonary and Critical Care Medicine, University of Alabama at Birmingham, 1900 University Blvd, THT 215, Birmingham, AL 35294-0006; e-mail: olman{at}pulm.dom.uab.edu

The fibrinolytic protease inhibitor plasminogen activator inhibitor type I (PAI-1) dramatically affects the outcome of lung inflammation and fibrosis in mice injured with either bleomycin or hyperoxia. Furthermore, during human and/or experimental lung injury, fibrin deposition colocalizes with high PAI-1, expressing alveolar mesenchymal cells in the remodeling alveolar matrix. As we have recently shown that PAI-1 is transcriptionally upregulated in lung fibroblasts by the major plasmin proteolytic fragment of fibrin (D-dimer), we undertook this study to determine the cis-acting elements and trans-acting factors that are responsible for fibrin D-dimer's action on PAI-1 transcription.

To study transcriptional activation, lung fibroblasts were transfected with a PAI-1 gene-lucifease reporter construct and stimulated with highly purified D-dimer. To determine the DNA binding proteins, the cells were stimulated with D-dimer and nuclear extracts were harvested for electrophoretic mobility shift assay (EMSA) or Northern blot analysis.

Basal PAI-1 transcriptional activity fell to 25% upon deletion of the -161 to -928 region of the PAI-1 gene and fell further to < 1% of the larger fragment upon deletion of the -161 to -48 region. In transiently transfected fibroblasts, D-dimer exposure (1 µM for 48 h) stimulated PAI-1 transcriptional activation fivefold in a manner that was entirely dependent on the presence of the 5' flanking region from -48 to -161 base pairs. By EMSA, D-dimer stimulation resulted in a specific, time (peak 45 min) and dose-dependent induction (peak sixfold at 100 nM to 1 µM) of c-fos/junD binding, as identified using specific antibodies, to the -66 to -45 base pairs DNA element of the PAI-1 gene. Mutation of the putative AP-1 binding site (-59 to -52) to a non-AP-1 binding mutant (as validated by EMSA) reduced basal transcriptional activity threefold and resulted in a complete loss of overexpressed c-fos inducibility of PAI-1 transcription in cotransfection assays.

These results indicate that the c-fos/junD binds to and transcriptionally activates the PAI-1 gene (by the -59 to -52 sequence) in response to fibrin-derived D-dimer in a dose- and time-dependent manner. As hyaline membranes and pulmonary fibrotic lesions contain fibrin and high PAI-1-expressing fibroblasts, these data provide further support for the importance of fibrin proteolytic fragments in the feedback regulation of PAI-1 expression and in the biology of matrix remodeling after acute lung injury.

Footnotes

This research was supported by a VA MERIT grant and by the National Heart, Lung, and Blood Institute.

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