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-kappaB (NF-kappaB), 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.


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


RESULTS
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.


CONCLUSIONS
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.
Pendergraft
William F
WF
Department of Medicine, Division of Nephrology and Hypertension, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7155, USA.
Rudolph
Earl H
EH
Falk
Ronald J
RJ
Jahn
Jennifer E
JE
Grimmler
Matthias
M
Hengst
Ludger
L
Jennette
J Charles
JC
Preston
Gloria A
GA
D017209:Apoptosis / Q000502:physiology*; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology; D016193:G1 Phase / Q000502:physiology; D053804:Granzymes; D006801:Humans; D007249:Inflammation / Q000378:metabolism; D053146:Myeloblastin; D016192:Resting Phase, Cell Cycle / Q000502:physiology; D012697:Serine Endopeptidases / Q000378:metabolism*; D013379:Substrate Specificity; D014471:Umbilical Veins / Q000166:cytology
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases; D053146:Myeloblastin; D020169:Caspases