Cleavage of the pro-survival protein intersectin-1s by granzyme B induces endothelial cell proliferation via activation of p38 MAPK and Elk-1 transcription factor

Endothelial cell (EC) phenotypic changes contribute to the various phases of inflammation during human disease and are key pathobiological players in lung vascular remodeling leading to chronic vascular disorders. In many inflammatory states such as rheumatoid arthritis, other connective tissue diseases, and some types of pulmonary hypertension, blood as well as tissue level of inflammatory cells, including T lymphocytes, is increased. The CD8+ cytotoxic T cells contain perforin and granzyme B (GrB) which are released from the cytotoxic granules to induce target cell death. The cytotoxic protease GrB can initiate ECs apoptosis by cleaving caspases and other proteins important for proliferation and cell survival. Using in vitro and in vivo approaches we now show that intersectin-1s (ITSN-1s), an essential protein for lung ECs survival, is cleaved by GrB at the sequence IDQD271K and generates a 28 kDa N-terminal cleavage product (GrB-EHITSN). Morphological analyses of cultured human Pulmonary Artery ECs (hPAECs) exposed to native human GrB show significant dysfunction and a gradual increase in apoptotic cell death; nonetheless, depending on GrB concentration some ECs may acquire an apoptotic resistant phenotype. Significantly, expression of myc-GrB-EHITSN in ECs causes marked increase in cell proliferation, as indicated by bromodeoxiuridine (BrdU) incorporation detected with anti BrdU Ab staining. Lysates and nuclear extracts prepared from control and myc-GrB-EHITSN transfected cells and subjected to Western blot analyses and ELISA indicate that GrB-EHITSN may be implicated in controlling cell proliferation via persistent activation of p38 MAPK and Elk-1 transcription factor. Altogether, our findings suggest that during inflammatory response, GrB-mediated proteolysis of ITSN-1s may provide an advantage for p38 MAPK signaling cascade and Elk-1 transcription factor favoring the selection of a hyper-proliferative EC phenotype.