Proteinase 3 sidesteps caspases and cleaves p21^{Waf1/Cip1/Sdi1} to induce endothelial cell apoptosis.Background. Emerging data raise possibilities of a complex and specific biologic role for leukocyte-derived proteases in substrate processing and in signaling pathways. Neutrophil proteinase 3 (PR3) is a caspase-like protease that enters endothelial cells, cleaves nuclear factor-κB (NF-κB), and induces sustained JNK activation, implying that the major cell cycle inhibitor p21 may be inactivated. Cleavage of p21 by caspase-3 is reported to be required for endothelial cell apoptosis. We hypothesized that PR3 may target p21.

Human umbilical vein endothelial cells (HUVEC) were treated with or without PR3 (5 μg/mL) from 0 hours or up to 8 hours, and analyzed for changes in cell cycle control proteins by immunoblotting, immunofluorescence and flow cytometry.

PR3 exposure resulted in cleavage of p21 between Thr⁸⁰ and Gly⁸¹, loss of nuclear p21 by cytoplasmic sequestration and depletion of p21 from cyclin/cyclin-dependent kinase (CDK) complexes. Examination of cyclins D and E, p53, Rb, and p27 revealed a largely nonproliferative expression profile. Cells arrested in G₁ were more susceptible to PR3 effects. We examined inflamed human colonic tissue and found a fragment similar in size to that generated by PR3 in HUVEC. Granzyme B, a T-cell homologue of PR3 that cleaves caspase substrates, also cleaves p21 between Asp⁶² and Phe⁶³. A reported substrate of granzyme B and caspases, Bid, is cleaved by PR3 signifying commonality of substrates among these proteases.

A theme is developing that the granulocyte protease, PR3, is an exogenous caspase-like molecule that can sidestep intracellular caspase functions at sites of inflammation.