Transforming growth factor alpha in arterioles: cell surface processing of its precursor by elastases.
Analysis of the transforming growth factor alpha (TGF alpha) cDNA predicts that the mature TGF alpha polypeptide is cleaved from the extracellular domain of its precursor, which is an integral membrane protein. Furthermore, the cleavage sites for the release of this mitogen are compatible with the participation of an elastaselike protease. We have immunohistochemically localized TGF alpha to the vascular smooth muscle cells in the arterioles. To investigate whether polymorphonuclear (PMN) leukocytic elastase, a blood-borne protease, could process the cell surface TGF alpha, NR6 cells were transfected with the rat TGF alpha cDNA. The cDNA encoded the entire open reading frame, and its expression was under the control of the mouse metallothionein I promoter. A cloned transfectant, termed 1B2, synthesized the TGF alpha precursor in a zinc-inducible manner, and the precursor was localized to the cell surface. Western blot (immunoblot) analysis indicated that treatment of the zinc-induced 1B2 cells with either PMN leukocytic or pancreatic elastase resulted in the release of the mature TGF alpha polypeptide. The released TGF alpha was bioactive, as it was capable of both competing with epidermal growth factor for binding to its receptor and stimulating [3H]thymidine incorporation in the mitogenic assay. Formaldehyde fixation of the 1B2 cells eliminated basal release of TGF alpha but allowed normal processing by both PMN leukocytic and pancreatic elastase to occur. However, human cathepsin G, bovine pancreatic alpha 1-chymotrypsin, collagenase, trypsin, subtilisin, and plasmin failed to release any detectable fragments of the TGF alpha precursor from the fixed cells. The location of TGF alpha in the arterioles and ability of PMN leukocytic elastase to process the membrane-bound TGF alpha precursor suggests a novel role for this elastase at the wound site.
Department of Clinical Biochemistry, University of Toronto, Banting and Best Diabetes Centre, Toronto General Hospital, Ontario, Canada.
S G
SG
Mueller
A J
AJ
Paterson
J E
JE
Kudlow
D000818:Animals; D001158:Arteries / Q000201:enzymology*; D001160:Arterioles / Q000201:enzymology*; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D066246:ErbB Receptors / Q000378:metabolism; D007124:Immunoenzyme Techniques; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D009504:Neutrophils / Q000201:enzymology; D010196:Pancreatic Elastase / Q000235:genetics*; D010903:Pituitary Gland, Anterior / Q000098:blood supply / Q000201:enzymology; D011499:Protein Processing, Post-Translational; D051381:Rats; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D014409:Tumor Necrosis Factor-alpha; D066246:ErbB Receptors; D010196:Pancreatic Elastase