An oxidative stress mechanism mediates chelerythrine-induced heparin-binding EGF-like growth factor ectodomain shedding.
Regulated shedding of cell surface proteins is a mechanism for rapid activation of autocrine and paracrine signaling. Here we report that chelerythrine, a protein kinase C (PKC) inhibitor that possesses a variety of biological functions, is a potent inducer of heparin-binding epidermal growth factor-like growth factor (HB-EGF) shedding from the cell surface. Chelerythrine induced a time- and dose-dependent shedding of an HB-EGF-alkaline phosphatase (HB-EGF-AP) fusion protein expressed in MC2 rat prostate epithelial cells. The soluble form of HB-EGF-AP bound to heparin and exhibited potent biological activity as measured by DNA synthesis assay. Chelerythrine-induced HB-EGF shedding was metalloproteinase-(MMP-) mediated because specific MMP antagonists inhibited shedding by > or =60%. Chelerythrine stimulated production of reactive oxygen species, and antioxidants prevented chelerythrine-induced HB-EGF shedding, suggesting that the production of intracellular peroxides is necessary for this event. Consistent with this possibility, antioxidant- and MMP-inhibitable shedding was also demonstrated when hydrogen peroxide was used as an inducer. Although JNK/SAPK and p38 MAPK pathways were activated by chelerythine, these signaling mechanisms were not required to mediate the shedding event. However, JNK signaling was involved in chelerythrine-stimulated apoptosis. Our results suggest that HB-EGF shedding induced by chelerythrine is mediated predominantly via the production of reactive oxygen species.
Kim
Jayoung
J
The Urological Diseases Research Center, Department of Urology, Children's Hospital Boston, Massachusetts 02115, USA.
Lin
Jianqing
J
Adam
Rosalyn M
RM
Lamb
Carolyn
C
Shively
Sharon Baughman
SB
Freeman
Michael R
MR
D000470:Alkaloids; D000818:Animals; D053119:Benzophenanthridines; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004815:Epidermal Growth Factor / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D008297:Male; D018384:Oxidative Stress; D010617:Phenanthridines / Q000494:pharmacology*; D011467:Prostate / Q000166:cytology / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D051381:Rats; D017382:Reactive Oxygen Species
D000470:Alkaloids; D053119:Benzophenanthridines; D004791:Enzyme Inhibitors; C587885:Hbegf protein, rat; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D010617:Phenanthridines; D017382:Reactive Oxygen Species; D004815:Epidermal Growth Factor; C016299:chelerythrine; D011493:Protein Kinase C