Proteolytic cleavage of granulocyte colony-stimulating factor and its receptor by neutrophil elastase induces growth inhibition and decreased cell surface expression of the granulocyte colony-stimulating factor receptor.
Neutrophil elastase (NE) is a serine protease stored in the primary granules of neutrophils that proteolytically cleaves multiple cytokines and cell surface proteins on release from activated neutrophils. Recent reports of mutations in the gene encoding this enzyme in some patients with neutropenic syndromes prompted us to investigate whether granulocyte colony-stimulating factor (G-CSF) and its receptor (G-CSFR) are also substrates for NE. To further address this, we examined the effect of NE on G-CSF and the G-CSFR both in solution and on intact cells. Incubation of recombinant G-CSF or a G-CSFR form corresponding to its extracellular domain with purified NE resulted in rapid proteolytic cleavage of both proteins. Addition of NE to tissue culture medium or pretreatment of G-CSF with NE before its addition to media suppressed the growth of G-CSF-responsive cells. NE also cleaved the G-CSFR on the surface of intact cells resulting in a time-dependent reduction in cell surface expression of the G-CSFR. Notably, decreased G-CSFR surface expression resulting from treatment of cells with NE was also associated with a reduction in cell viability and proliferation in response to G-CSF. These results are the first to demonstrate that G-CSF and G-CSFR are proteolytically cleaved by NE and that NE-induced degradation of these proteins correlates with a reduction in the biologic activity of the cytokine and a decrease in the signaling function of the receptor because of decreased G-CSFR surface expression. These findings provide additional insights into mechanisms by which G-CSF/G-CSFR interactions may be modulated.
Bone Marrow Transplant Program, The Arthur G James Cancer Hospital and Richard J Solove Research Institute, The Ohio State University College of Medicine and Public Health, Columbus, Ohio 43210, USA.
Melissa G
MG
Hunter
Lawrence J
LJ
Druhan
Pam R
PR
Massullo
Belinda R
BR
Avalos
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D016179:Granulocyte Colony-Stimulating Factor / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D019272:Leukocyte Elastase / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000378:metabolism*; D014162:Transfection
D016188:Receptors, Granulocyte Colony-Stimulating Factor; D016179:Granulocyte Colony-Stimulating Factor; D019272:Leukocyte Elastase